78 results on '"Linda K. Nicholson"'
Search Results
2. The IL-33-PIN1-IRAK-M axis is critical for type 2 immunity in IL-33-induced allergic airway inflammation
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Morris Nechama, Jeahoo Kwon, Shuo Wei, Adrian Tun-Kyi, Robert S. Welner, Iddo Z. Ben-Dov, Mohamed S. Arredouani, John M. Asara, Chun-Hau Chen, Cheng-Yu Tsai, Kyle F. Nelson, Koichi S Kobayashi, Elliot Israel, Xiao Zhen Zhou, Linda K. Nicholson, and Kun Ping Lu
- Subjects
Science - Abstract
Abstract Interleukin 33 (IL-33) is among the earliest-released cytokines in response to allergens that orchestrate type 2 immunity. The prolyl cis-trans isomerase PIN1 is known to induce cytokines for eosinophil survival and activation by stabilizing cytokines mRNAs, but the function of PIN1 in upstream signaling pathways in asthma is unknown. Here we show that interleukin receptor associated kinase M (IRAK-M) is a PIN1 target critical for IL-33 signaling in allergic asthma. NMR analysis and docking simulations suggest that PIN1 might regulate IRAK-M conformation and function in IL-33 signaling. Upon IL-33-induced airway inflammation, PIN1 is activated for binding with and isomerization of IRAK-M, resulting in IRAK-M nuclear translocation and induction of selected proinflammatory genes in dendritic cells. Thus, the IL-33-PIN1-IRAK-M is an axis critical for dendritic cell activation, type 2 immunity and IL-33 induced airway inflammation.
- Published
- 2018
- Full Text
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3. Lineshape Analysis as a Tool for Probing Functional Motions at Biological Interfaces
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Soumya De, Alexander I. Greenwood, L. Andrea Acevedo, Nathan E. Korson, and Linda K. Nicholson
- Abstract
The analysis of peak lineshapes in two-dimensional NMR spectra acquired during titration experiments is a powerful technique for quantitative studies of biological interfaces. The theoretical basis for how the thermodynamics (equilibrium populations) and kinetics (rate constants) of a multi-state equilibrium are encoded in the lineshape of an NMR peak is presented. Insights on how an exchange process influences the observed magnetization, how to construct the rate matrix for a given reaction scheme, and the origins of the governing lineshape equation are included. Applications of lineshape analysis to two-state binding and to four-state peptidyl prolyl cis–trans isomerization reactions involved in Alzheimer's disease and lateral root development in rice are presented. These studies illustrate how lineshape analysis of NMR titration experiments provides key mechanistic details for understanding the role of biological interfaces. Importantly, the quantitative model obtained from observations of an exchange reaction in the NMR tube enables predictions of activity to be scaled to cellular concentrations. This method is highly complementary to more recently developed NMR relaxation techniques. The application of lineshape analysis and relaxation methods to the same samples could enable quantitative characterization of even more complex multi-state systems.
- Published
- 2022
4. Solution structure of the phosphatidylinositol 3-phosphate binding domain from theLegionellaeffector SetA
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Wendy H.J. Beck, Thais A. Enoki, Xiaochun Wu, Qing Zhang, Linda K. Nicholson, Robert E. Oswald, and Yuxin Mao
- Abstract
Legionella pneumophilais a facultative intracellular pathogen that causes Legionnaires’ disease or Pontiac fever in humans upon accidental inhalation ofLegionella-contaminated aerosols. During infection,L. pneumophilasecretes more than 300 effectors into the host for the biogenesis of a replication-permissive niche, known as theLegionella containingvacuole (LCV). Among these, a large number of effectors harbor protein domains that recognize specific phosphoinositide (PI) lipids and mediate the anchoring of these effectors to the surface of LCV or other host membrane-bound organelles. TheLegionellaeffector SetA contains a unique C-terminal domain (SetA-CTD) that has been shown to specifically bind with phosphatidylinositol-3-phosphate (PI(3)P) and target SetA to endosomes and LCVs. Here, we report the NMR solution structure of SetA-CTD, which mainly comprises a four α-helix bundle. The structure reveals a basic pocket at one end of the α-helix bundle for PI(3)P binding and two hydrophobic loops for membrane insertion. Mutations of key residues involved in lipid binding result in the loss of SetA in membrane association and endosomal localization. Structural comparison with other PI(3)P-binding domains highlights a general theme applied by multiple families of phosphoinositide-binding domains across species.
- Published
- 2022
5. Quantification of reaction cycle parameters for an essential molecular switch in an auxin-responsive transcription circuit in rice
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Linda K. Nicholson, Lucila A. Acevedo, and Jeahoo Kwon
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Models, Molecular ,0106 biological sciences ,0301 basic medicine ,Transcription, Genetic ,Isomerase ,Genes, Plant ,01 natural sciences ,Catalysis ,Substrate Specificity ,03 medical and health sciences ,Isomerism ,Plant Growth Regulators ,Auxin ,Transcription (biology) ,Nuclear Magnetic Resonance, Biomolecular ,Cyclophilin ,Plant Proteins ,chemistry.chemical_classification ,Molecular switch ,Regulation of gene expression ,Multidisciplinary ,Indoleacetic Acids ,Lateral root ,Oryza ,Biological Sciences ,Kinetics ,030104 developmental biology ,chemistry ,Biophysics ,Thermodynamics ,Isomerization ,010606 plant biology & botany - Abstract
Protein-based molecular switches play critical roles in biological processes. The importance of the prolyl cis − trans switch is underscored by the ubiquitous presence of peptidyl prolyl isomerases such as cyclophilins that accelerate the intrinsically slow isomerization rate. In rice, a tryptophan−proline (W-P) cis − trans switch in transcription repressor protein OsIAA11 along with its associated cyclophilin LRT2 are essential components in a negative feedback gene regulation circuit that controls lateral root initiation in response to the plant hormone auxin. Importantly, no quantitative characterizations of the individual (microscopic) thermodynamic and kinetic parameters for any cyclophilin-catalyzed W-P isomerization have been reported. Here we present NMR studies that determine and independently validate these parameters for LRT2 catalysis of the W-P motif in OsIAA11, providing predictive power for understanding the role of this switch in the auxin-responsive circuit and the resulting lateral rootless phenotype in rice. We show that the observed isomerization rate is linearly dependent on LRT2 concentration but is independent of OsIAA11 concentration over a wide range, and LRT2 is optimally tuned to maintain OsIAA11 at its cis − trans equilibrium to supply the slower downstream cis- specific proteasomal degradation with maximal OsIAA11 substrate. This indicates that accelerating the LRT2-catalyzed isomerization would not accelerate OsIAA degradation, whereas decreasing this rate via targeted mutation could reveal relationships between circuit dynamics and lateral root development. Moreover, we show that sequences flanking the highly conserved Aux/IAA W-P motif do not impact LRT2 catalysis, suggesting that the parameters determined here are broadly applicable across highly conserved cyclophilins and their Aux/IAA targets.
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- 2019
6. The IL-33-PIN1-IRAK-M axis is critical for type 2 immunity in IL-33-induced allergic airway inflammation
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Xiao Zhen Zhou, Adrian Tun Kyi, John M. Asara, Iddo Z. Ben-Dov, Elliot Israel, Koichi Kobayashi, Mohamed S. Arredouani, Linda K. Nicholson, Kun Ping Lu, Morris Nechama, Chun-Hau Chen, Jeahoo Kwon, Shuo Wei, Robert S. Welner, Kyle F. Nelson, and Cheng-Yu Tsai
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Male ,0301 basic medicine ,General Physics and Astronomy ,Mice ,lcsh:Science ,Lung ,Mice, Knockout ,Immunity, Cellular ,Multidisciplinary ,Chemistry ,3. Good health ,Molecular Docking Simulation ,Interleukin-1 Receptor-Associated Kinases ,medicine.anatomical_structure ,PIN1 ,Interleukin receptor ,Female ,Signal transduction ,Bronchoalveolar Lavage Fluid ,Signal Transduction ,Adult ,Science ,Primary Cell Culture ,Bronchial Provocation Tests ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Proinflammatory cytokine ,Young Adult ,03 medical and health sciences ,Th2 Cells ,Protein Domains ,Immunity ,medicine ,Animals ,Humans ,Antigens, Dermatophagoides ,General Chemistry ,Dendritic cell ,Eosinophil ,Interleukin-33 ,Asthma ,Eosinophils ,NIMA-Interacting Peptidylprolyl Isomerase ,Interleukin 33 ,Disease Models, Animal ,HEK293 Cells ,030104 developmental biology ,Immunology ,lcsh:Q - Abstract
Interleukin 33 (IL-33) is among the earliest-released cytokines in response to allergens that orchestrate type 2 immunity. The prolyl cis-trans isomerase PIN1 is known to induce cytokines for eosinophil survival and activation by stabilizing cytokines mRNAs, but the function of PIN1 in upstream signaling pathways in asthma is unknown. Here we show that interleukin receptor associated kinase M (IRAK-M) is a PIN1 target critical for IL-33 signaling in allergic asthma. NMR analysis and docking simulations suggest that PIN1 might regulate IRAK-M conformation and function in IL-33 signaling. Upon IL-33-induced airway inflammation, PIN1 is activated for binding with and isomerization of IRAK-M, resulting in IRAK-M nuclear translocation and induction of selected proinflammatory genes in dendritic cells. Thus, the IL-33-PIN1-IRAK-M is an axis critical for dendritic cell activation, type 2 immunity and IL-33 induced airway inflammation., IL-33 orchestrates type 2 immunity in allergic asthma. Here the authors show, using biochemical, structural and patient data, that upon IL-33 or allergic challenge, the isomerase Pin1 modifies IRAK-M to control the production of pro-inflammatory cytokines in the setting of airway inflammation.
- Published
- 2018
7. A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin
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Linda K. Nicholson, Alexander I. Greenwood, and Lucila A. Acevedo
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Protein domain ,macromolecular substances ,Biochemistry ,Article ,Zyxin ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,EVH1 domain ,Humans ,Amino Acid Sequence ,Cytoskeleton ,biology ,Microfilament Proteins ,Vasodilator-stimulated phosphoprotein ,Signal transducing adaptor protein ,Vinculin ,Phosphoproteins ,Cell biology ,030104 developmental biology ,Phosphoprotein ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Cell Adhesion Molecules ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase with roles in the control of cell shape and cell migration. Through interaction with the cytoskeletal adaptor protein Zyxin, VASP can localize to damaged stress fibers where it serves to repair and reinforce these structures. VASP localization is mediated by its N-terminal Ena/VASP homology (EVH1) domain, which binds to the (W/F)PxφP motif (most commonly occurring as FPPPP) found in cytoskeletal proteins such as vinculin, lamellipodin, and Zyxin. Sequentially close clusters of four or five of these motifs frequently occur, as in the proline rich region of Zyxin with four such motifs. This suggests that tetrameric VASP might bind very tightly to Zyxin through avidity, with all four EVH1 domains binding to a single Zyxin molecule. Here, quantitative nuclear magnetic resonance titration analysis reveals a dominant bivalent 1:1 (Zyxin:EVH1) interaction between the Zyxin proline rich region and the VASP EVH1 domain that utilizes the EVH1 canonical binding site and a novel secondary binding site on the opposite face of the EVH1 domain. We further show that binding to the secondary binding site is specifically inhibited by mutation of VASP EVH1 domain residue Y39 to E, which mimics Abl-induced phosphorylation of Y39. On the basis of these findings, we propose a model in which phosphorylation of Y39 acts as a stoichiometry switch that governs binding partner selection by the constitutive VASP tetramer. These results have broader implications for other multivalent VASP EVH1 domain binding partners and for furthering our understanding of the role of Y39 phosphorylation in regulating VASP localization and cellular function.
- Published
- 2017
8. Neighboring phosphoSer‐Pro motifs in the undefined domain of <scp>IRAK</scp> 1 impart bivalent advantage for Pin1 binding
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Jeahoo Kwon, Linda K. Nicholson, Monique J. Rogals, Alexander I. Greenwood, and Kun Ping Lu
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Models, Molecular ,0301 basic medicine ,Magnetic Resonance Spectroscopy ,Proline ,Stereochemistry ,Amino Acid Motifs ,Biosensing Techniques ,Binding, Competitive ,Biochemistry ,Article ,Bivalent (genetics) ,Interleukin-1 receptor-associated kinase 1 ,WW domain ,03 medical and health sciences ,Protein Domains ,Catalytic Domain ,Serine ,Prolyl isomerase ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,IRAK1 ,Cell Biology ,Affinities ,NIMA-Interacting Peptidylprolyl Isomerase ,Kinetics ,Interferometry ,Interleukin-1 Receptor-Associated Kinases ,030104 developmental biology ,Models, Chemical ,biology.protein ,PIN1 ,Peptides ,Protein Binding - Abstract
The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, as many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein interleukin-1 receptor-associated kinase-1 (IRAK1). The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly phosphorylated peptides showed that individual motifs displayed weak affinities (> 100 μm) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 and 24 μm for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1.
- Published
- 2016
9. Tuning a timing device that regulates lateral root development in rice
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Linda K. Nicholson, Justin M Williams, Nathan E Korson, and Lucila A. Acevedo
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0301 basic medicine ,Mutant ,Isomerase ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Plant Roots ,Article ,03 medical and health sciences ,Isomerism ,Transcription (biology) ,Auxin ,Peptide bond ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Plant Proteins ,chemistry.chemical_classification ,Indoleacetic Acids ,Chemistry ,Protein Stability ,Lateral root ,food and beverages ,Oryza ,Peptidylprolyl Isomerase ,Cis trans isomerization ,0104 chemical sciences ,030104 developmental biology ,PPIB ,Biophysics ,Mutant Proteins - Abstract
Peptidyl Prolyl Isomerases (PPIases) accelerate cis-trans isomerization of prolyl peptide bonds. In rice, the PPIase LRT2 is essential for lateral root initiation. LRT2 displays in vitro isomerization of a highly conserved W-P peptide bond ((104)W-P(105)) in the natural substrate OsIAA11. OsIAA11 is a transcription repressor that, in response to the plant hormone auxin, is targeted to ubiquitin-mediated proteasomal degradation via specific recognition of the cis isomer of its (104)W-P(105) peptide bond. OsIAA11 controls transcription of specific genes, including its own, that are required for lateral root development. This auxin-responsive negative feedback circuit governs patterning and development of lateral roots along the primary root. The ability to tune LRT2 activity via mutagenesis is crucial for understanding and modeling the role of this bimodal switch in the auxin circuit and lateral root development. We present characterization of the thermal stability and isomerization rates of several LRT2 mutants acting on the OsIAA11 substrate. The thermally stable mutants display activities lower than that of wild-type (WT) LRT2. These include binding diminished but catalytically active P125K, binding incompetent W128A, and binding capable but catalytically incompetent H133Q mutations. Additionally, LRT2 homologs hCypA from human, TaCypA from Triticum aestivum (wheat) and PPIB from E. coli were shown to have 110%, 50% and 60% of WT LRT2 activity on the OsIAA11 substrate. These studies identify several thermally stable LRT2 mutants with altered activities that will be useful for establishing relationships between cis-trans isomerization, auxin circuit dynamics, and lateral root development in rice.
- Published
- 2019
10. Structure and Function in Antimicrobial Piscidins: Histidine Position, Directionality of Membrane Insertion, and pH-dependent Permeabilization
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Richard W. Pastor, Linda K. Nicholson, Vitalii Silin, Jolita Seckute, Janet Hammer, Mirco Sorci, Nedzada Smajic, Alexander I. Greenwood, Jorge I. Hernandez, B. Scott Perrin, Jack Blazyk, Riqiang Fu, Akritee Shrestha, Myriam Cotten, Kimberly A. Bogardus, Mihaela Mihailescu, and Georges Belfort
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Fish Proteins ,Antimicrobial peptides ,Lipid Bilayers ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,Permeability ,Surface-Active Agents ,Colloid and Surface Chemistry ,Amphiphile ,Animals ,Histidine ,Amino Acid Sequence ,Surface plasmon resonance ,Lipid bilayer ,Fluorescent Dyes ,Chemistry ,Bilayer ,Fishes ,Phosphatidylglycerols ,General Chemistry ,Quartz crystal microbalance ,Hydrogen-Ion Concentration ,Fluoresceins ,0104 chemical sciences ,Membrane ,Biophysics ,Phosphatidylcholines ,Antimicrobial Cationic Peptides - Abstract
Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.
- Published
- 2019
11. 1H, 13C and 15N NMR assignments of cyclophilin LRT2 (OsCYP2) from rice
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Linda K. Nicholson and Lucila A. Acevedo
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0301 basic medicine ,chemistry.chemical_classification ,Oryza sativa ,Stereochemistry ,food and beverages ,Biochemistry ,Article ,Amino acid ,03 medical and health sciences ,030104 developmental biology ,Enzyme ,Heteronuclear molecule ,chemistry ,Structural Biology ,Isomerization ,Protein secondary structure ,Cyclophilin ,Function (biology) - Abstract
Cyclophilins are enzymes that catalyze the isomerization of a prolyl–peptide bond and are found in both prokaryotes and eukaryotes. LRT2 (also known as OsCYP2) is a cyclophilin in rice (Oryza sativa), that has importance in lateral root development and stress tolerance. LRT2 is 172 amino acids long and has a molecular weight of 18.3 kDa. Here, we report the backbone and sidechain resonance assignments of 1H, 13C, 15N in the LRT2 protein using several 2D and 3D heteronuclear NMR experiments at pH 6.7 and 298 K. Our chemical shift data analysis predicts a secondary structure like the cytosolic wheat cyclophilin TaCypA-1 with 87.7% sequence identity. These assignments will be useful for further analysis in the NMR studies for function and structure of this enzyme.
- Published
- 2018
12. Mechanism of midline defect-causing mutation P151L in MID1 revealed
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Linda K. Nicholson
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0301 basic medicine ,Male ,biology ,Hypertelorism ,Chemistry ,Phosphatase ,Opitz G Syndrome ,Regulator ,Ubiquitination ,Cell Biology ,Protein phosphatase 2 ,Biochemistry ,Midline defects ,Cell biology ,Ubiquitin ligase ,03 medical and health sciences ,030104 developmental biology ,Mutation (genetic algorithm) ,Mutation ,biology.protein ,Microtubule Proteins ,Humans ,Protein Phosphatase 2 ,Molecular Biology ,Transcription Factors - Abstract
The P151L mutation in the B-box1 domain of MID1 causes midline defects in X-linked Opitz G Syndrome. MID1 is known to be a key regulator of phosphatase PP2A through formation of a complex with its catalytic (PP2Ac) and regulatory (α4) subunits. Wright et al. show that this mutation retains B-box1 domain structure and E3 ligase activity (star) but blocks interaction with α4, indicating disruption of the MID1-α4-PP2Ac complex.
- Published
- 2017
13. Cyclic cis-locked phospho-dipeptides reduce entry of AβPP into amyloidogenic processing pathway
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Carolyn L. Fisher, Lucila A. Acevedo, Linda K. Nicholson, Ross J. Resnick, Frank C. Schroeder, Soumya De, and Kun Ping Lu
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0301 basic medicine ,Blotting, Western ,Cleavage (embryo) ,Transfection ,Article ,Biological pathway ,03 medical and health sciences ,chemistry.chemical_compound ,Amyloid beta-Protein Precursor ,Cell Line, Tumor ,mental disorders ,Peptide bond ,Aspartic Acid Endopeptidases ,Humans ,Enzyme Inhibitors ,Protein precursor ,Dipeptide ,biology ,Dose-Response Relationship, Drug ,Molecular Structure ,Chemistry ,General Neuroscience ,General Medicine ,Dipeptides ,Cell biology ,Psychiatry and Mental health ,Clinical Psychology ,030104 developmental biology ,Neuroprotective Agents ,Cytoplasm ,Culture Media, Conditioned ,biology.protein ,Geriatrics and Gerontology ,Amyloid Precursor Protein Secretases ,Amyloid precursor protein secretase - Abstract
The cis/trans isomerization of X-Pro peptide bonds in proteins in some instances acts as a molecular switch in biological pathways. Our prior work suggests that the cis isomer of the phospho-Thr668-Pro669 motif, located in the cytoplasmic domain of the amyloid-β precursor protein (AβPP), is correlated with an increase in amyloidogenic processing of AβPP and production of amyloid beta (Aβ), the neurotoxic peptide fragment in Alzheimer’s disease (AD). We designed a 100% cis-locked cyclic dipeptide composed of cyclized phospho-Thr-Pro (pCDP) as a mimic for this putative pathological conformation, and three phosphate-blocked derivatives (pCDP-diBzl, pCDP-Bzl, and pCDP-diPOM). Two H4 neuroglioma cell lines were established as AD cell models for use in testing these compounds: H4-AβPP695 for stable overexpression of wild-type AβPP695, and H4-BACE1 for stable overexpression of β-site AβPP Cleaving Enzyme-1 (BACE1). The level of the secreted AβPP fragment resulting from BACE1 activity, sAβPPβ, served as a key proxy for amyloidogenic processing, since cleavage of AβPP by BACE1 is a requisite first step in Aβ production. Of the compounds tested, pCDP-diBzl decreased sAβPPβ levels in both cell lines, while pCDP-diPOM decreased sAβPPβ levels in only H4-BACE1 cells, all with similar dose-dependences and patterns of proteolytic AβPP fragments. Enzymatic assays showed that none of the pCDP derivatives directly inhibit BACE1 catalytic activity. These results suggest a model in which pCDP-diBzl and pCDP-diPOM act at a common point to inhibit entry of AβPP into the amyloidogenic AβPP processing pathway but through different targets, and provide important insights for the development of novel AD therapeutics. Keywords: Amyloid beta-Protein Precursor, Alzheimer’s Disease, cyclic dipeptides, diketopiperazine, phosphorylated Thr668.
- Published
- 2017
14. Isomerase-Catalyzed Binding of Interleukin-1 Receptor-Associated Kinase 1 to the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein
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Linda K. Nicholson, Alexander I. Greenwood, and Jeahoo Kwon
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Kinase ,Microfilament Proteins ,Vasodilator-stimulated phosphoprotein ,macromolecular substances ,Isomerase ,Biology ,Crystallography, X-Ray ,Phosphoproteins ,Biochemistry ,Catalysis ,Protein Structure, Tertiary ,Cell biology ,Interleukin-1 Receptor-Associated Kinases ,EVH1 domain ,Phosphoprotein ,Humans ,Peptide bond ,Isomerases ,Cytoskeleton ,Protein kinase A ,Cell Adhesion Molecules ,Protein Binding - Abstract
Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP–IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 μM). We further show that this motif adopts distinct cis and trans isomers for the Trp168–Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168–Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes.
- Published
- 2014
15. TCR Scanning of Peptide/MHC through Complementary Matching of Receptor and Ligand Molecular Flexibility
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William F. Hawse, Brian M. Baker, Soumya De, K. Chrisopher Garcia, Linda K. Nicholson, David M. Kranz, Alexander I. Greenwood, Evgeni L. Kovrigin, and Jaroslav Zajicek
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chemistry.chemical_classification ,biology ,Ligand ,Immunology ,T-cell receptor ,hemic and immune systems ,chemical and pharmacologic phenomena ,Peptide ,Complementarity determining region ,Plasma protein binding ,Major histocompatibility complex ,Protein structure ,chemistry ,Biochemistry ,biology.protein ,Biophysics ,Immunology and Allergy ,Heteronuclear single quantum coherence spectroscopy - Abstract
Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear magnetic resonance to study TCR–pMHC binding, examining recognition of the QL9/H-2Ld complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3β loop remains mobile within the TCR–pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3β is also mobile in the free pMHC and in the TCR–pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3β and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR–pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with sufficient stability to enable T cell signaling.
- Published
- 2014
16. Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity
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Katherine A. Fitzgerald, John M. Asara, Linda K. Nicholson, Tae Ho Lee, George C. Tsokos, Greg Finn, Adrian Tun-Kyi, Michael Nowak, Elliot Israel, Alexander I. Greenwood, Mark A. Exley, Kun Ping Lu, and Xiaoxia Li
- Subjects
Receptor complex ,Magnetic Resonance Spectroscopy ,Immunology ,Immunoblotting ,Biology ,Adaptive Immunity ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Immune system ,Immunology and Allergy ,Animals ,Phosphorylation ,030304 developmental biology ,Peptidylprolyl isomerase ,Mice, Knockout ,0303 health sciences ,Toll-like receptor ,Innate immune system ,Reverse Transcriptase Polymerase Chain Reaction ,Toll-Like Receptors ,TLR7 ,Dendritic Cells ,Interferon-beta ,Peptidylprolyl Isomerase ,Acquired immune system ,Immunity, Innate ,3. Good health ,Cell biology ,Mice, Inbred C57BL ,NIMA-Interacting Peptidylprolyl Isomerase ,Interleukin-1 Receptor-Associated Kinases ,030220 oncology & carcinogenesis ,PIN1 ,RNA ,Interferon Regulatory Factor-1 ,Signal Transduction - Abstract
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.
- Published
- 2011
17. The single kinin receptor signals to separate and independent physiological pathways in Malpighian tubules of the yellow fever mosquito
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Stephen A. Schepel, Austin William Blum, Jason D. Yang, Tiffany Sou, Kenneth Lau, Felix Tiburcy, Ronald J. Nachman, Klaus W. Beyenbach, Linda K. Nicholson, Jeremy T. Miyauchi, Peter M. Piermarini, and Andrew J. Fox
- Subjects
medicine.medical_specialty ,Malpighian tubule system ,animal structures ,Protein Conformation ,Physiology ,Kinins ,Aedes aegypti ,Malpighian Tubules ,Membrane Potentials ,Receptors, G-Protein-Coupled ,Structure-Activity Relationship ,Chlorides ,Aedes ,Physiology (medical) ,biology.animal ,Internal medicine ,Electric Impedance ,medicine ,Animals ,Receptor ,Cockroach ,biology ,Sodium ,Yellow fever ,Epithelial Cells ,Articles ,Kinin ,biology.organism_classification ,medicine.disease ,Body Fluids ,Cell biology ,Kinetics ,Endocrinology ,Mechanism of action ,Potassium ,Insect Proteins ,Yellow fever virus ,medicine.symptom ,circulatory and respiratory physiology ,Signal Transduction - Abstract
In the past, we have used the kinins of the cockroach Leucophaea (the leucokinins) to evaluate the mechanism of diuretic action of kinin peptides in Malpighian tubules of the yellow fever mosquito Aedes aegypti . Now using the kinins of Aedes (the aedeskinins), we have found that in isolated Aedes Malpighian tubules all three aedeskinins (1 μM) significantly 1) increased the rate of fluid secretion (V̇S), 2) hyperpolarized the basolateral membrane voltage (Vbl), and 3) decreased the input resistance (Rin) of principal cells, consistent with the known increase in the Cl−conductance of the paracellular pathway in Aedes Malpighian tubules. Aedeskinin-III, studied in further detail, significantly increased V̇Swith an EC50of 1.5 × 10−8M. In parallel, the Na+concentration in secreted fluid significantly decreased, and the K+concentration significantly increased. The concentration of Cl−remained unchanged. While the three aedeskinins triggered effects on Vbl, Rin, and V̇S, synthetic kinin analogs, which contain modifications of the COOH-terminal amide pentapeptide core sequence critical for biological activity, displayed variable effects. For example, kinin analog 1578 significantly stimulated V̇Sbut had no effect on Vbland Rin, whereas kinin analog 1708 had no effect on V̇Sbut significantly affected Vbland Rin. These observations suggest separate signaling pathways activated by kinins. One triggers the electrophysiological response, and the other triggers fluid secretion. It remains to be determined whether the two signaling pathways emanate from a single kinin receptor via agonist-directed signaling or from a differentially glycosylated receptor. Occasionally, Malpighian tubules did not exhibit a detectable response to natural and synthetic kinins. Hypothetically, the expression of the kinin receptor may depend on developmental, nutritional, and/or reproductive signals.
- Published
- 2010
18. Tuning a Prolyl Cis/Trans Molecular Switch that Regulates lateral Root Development in Rice
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Linda K. Nicholson and Lucila A. Acevedo
- Subjects
Molecular switch ,Chemistry ,Lateral root ,Biophysics ,Cis–trans isomerism - Published
- 2018
19. Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin
- Author
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Yi-Pin Lin, Linda K. Nicholson, Dae-Won Lee, Yogendra Sharma, Yung-Fu Chang, and Sean P. McDonough
- Subjects
Molecular Sequence Data ,Immunoglobulins ,Enzyme-Linked Immunosorbent Assay ,Plasma protein binding ,Biochemistry ,Bacterial Adhesion ,Extracellular matrix ,Bacterial Proteins ,Tropoelastin ,Leptospira ,Laminin ,Humans ,Biotinylation ,Amino Acid Sequence ,Lung ,Molecular Biology ,Aorta ,Skin ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Cell Biology ,Cells, Immobilized ,biology.organism_classification ,Molecular biology ,Elastin ,Fibronectin ,Microscopy, Fluorescence ,Protein Structure and Folding ,Mutation ,biology.protein ,Leptospira interrogans ,Protein Binding - Abstract
Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7'-8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17-27HTE) as determined by ELISA (LigBCon4, K(D) = 0.50 microm; LigBCen7'-8, K(D) = 0.82 microm; LigBCen9, K(D) = 1.54 microm; and LigBCen12, K(D) = 0.73 microm). The interaction of LigBCon4 and 17-27HTE was further confirmed by steady state fluorescence spectroscopy (K(D) = 0.49 microm) and ITC (K(D) = 0.54 microm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17-27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.
- Published
- 2009
20. The N-terminal region of Pseudomonas type III effector AvrPtoB elicits Pto-dependent immunity and has two distinct virulence determinants
- Author
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Ping He, Fangming Xiao, Linda K. Nicholson, Jennifer E. Dawson, Gregory B. Martin, Robert B. Abramovitch, and Jen Sheen
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Genetics ,Mutation ,Structural similarity ,Effector ,Mutagenesis ,Virulence ,Cell Biology ,Plant Science ,Biology ,medicine.disease_cause ,biology.organism_classification ,Structural biology ,Arabidopsis ,Pseudomonas syringae ,medicine - Abstract
Summary Resistance to bacterial speck disease in tomato is activated by the physical interaction of the host Pto kinase with either of the sequence-dissimilar type III effector proteins AvrPto or AvrPtoB (HopAB2) from Pseudomonas syringae pv. tomato. Pto-mediated immunity requires Prf, a protein with a nucleotide-binding site and leucine-rich repeats. The N-terminal 307 amino acids of AvrPtoB were previously reported to interact with the Pto kinase, and we show here that this region (AvrPtoB 1-307) is sufficient for eliciting Pto/Prf-dependent immunity against P. s. pv. tomato. AvrPtoB 1-307 was also found to be sufficient for a virulence activity that enhances ethylene production and increases growth of P. s. pv. tomato and severity of speck disease on susceptible tomato lines lacking either Pto or Prf. Moreover, we found that residues 308‐387 of AvrPtoB are required for the previously reported ability of AvrPtoB to suppress pathogen-associated molecular patternsinduced basal defenses in Arabidopsis. Thus, the N-terminal region of AvrPtoB has two structurally distinct domains involved in different virulence-promoting mechanisms. Random and targeted mutagenesis identified five tightly clustered residues in AvrPtoB 1-307 that are required for interaction with Pto and for elicitation of immunity to P. s. pv. tomato. Mutation of one of the five clustered residues abolished the ethylene-associated virulence activity of AvrPtoB 1-307. However, individual mutations of the other four residues, despite abolishing interaction with Pto and avirulence activity, had no effect on AvrPtoB1-307 virulence activity. None of these mutations affected the basal defense-suppressing activity of AvrPtoB1-387. Based on sequence alignments, estimates of helical propensity, and the previously reported structure of AvrPto, we hypothesize that the Ptointeracting domains of AvrPto and AvrPtoB 1-307 have structural similarity. Together, these data support a model in which AvrPtoB 1-307 promotes ethylene-associated virulence by interaction not with Pto but with another unknown host protein.
- Published
- 2007
21. Unexpected VASP-EVH1 Interactions with Irak1 and Zyxin: Phosphorylation and Bivalent Binding
- Author
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Linda K. Nicholson, Alexander I. Greenwood, Kun P. Lu, Jeahoo Kwon, and Lucila A. Acevedo
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Regulation of gene expression ,EVH1 domain ,Chemistry ,Biophysics ,Phosphorylation ,macromolecular substances ,Subcellular localization ,Cytoskeleton ,Sequence motif ,Actin ,Zyxin ,Cell biology - Abstract
VASP is an actin polymerization regulatory protein, and is a founding member of the Ena/VASP family of proteins. The EVH1 domain, a conserved component of the Ena/VASP family, localizes these proteins to sites of high cytoskeletal dynamics through its selective binding to the sequence motif (F/W)PxhP, where x is any residue and h is an aliphatic residue. Our NMR studies of VASP-EVH1 interactions with the innate immunity signaling protein IRAK1 and with the cytoskeletal protein zyxin have revealed an unexpected mechanism of regulation and an unexpected mode of binding, respectively. These results provide important insights into the transient subcellular localization of VASP in response to innate immunity signaling, and to a novel binding mode that could provide an advantage for specific partners that contain sequential binding motifs.
- Published
- 2015
- Full Text
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22. The Solution Structure of Type III Effector Protein AvrPto Reveals Conformational and Dynamic Features Important for Plant Pathogenesis
- Author
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Jennifer Wulf, Gregory B. Martin, Amr Fahmy, Pete E. Pascuzzi, and Linda K. Nicholson
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Models, Molecular ,0106 biological sciences ,Protein Folding ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Molecular Sequence Data ,Pseudomonas syringae ,Virulence ,Protein Serine-Threonine Kinases ,Biology ,01 natural sciences ,Structure-Activity Relationship ,03 medical and health sciences ,Protein structure ,Bacterial Proteins ,Solanum lycopersicum ,Structural Biology ,Secretion ,Amino Acid Sequence ,Molecular Biology ,Plant Proteins ,030304 developmental biology ,0303 health sciences ,Effector ,Omega loop ,Bacterial effector protein ,Cell biology ,Biochemistry ,Structural Homology, Protein ,Mutagenesis, Site-Directed ,Protein folding ,Protein Binding ,010606 plant biology & botany - Abstract
Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease of tomato, uses a type III secretion system (TTSS) to deliver effector proteins into the host cell. In resistant plants, the bacterial effector protein AvrPto physically interacts with the host Pto kinase and elicits antibacterial defense responses. In susceptible plants, which lack the Pto kinase, AvrPto acts as a virulence factor to promote bacterial growth. The solution structure of AvrPto reveals a functional core consisting of a three-helix bundle motif flanked by disordered N- and C-terminal tails. Residues required for Pto binding lie in a 19 residue Omega loop. Modeling suggests a hydrophobic patch involving the activation loop of Pto forms a contact surface with the AvrPto Omega loop and that helix packing mediates interactions between AvrPto and putative virulence targets Api2 and Api3. The AvrPto structure has a low stability that may facilitate chaperone-independent secretion by the TTSS.
- Published
- 2004
23. [Untitled]
- Author
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Linda K. Nicholson, Jason D. Gans, and Norma H. Pawley
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Quantitative Biology::Biomolecules ,Mathematical optimization ,Observational error ,Chemistry ,Relaxation (NMR) ,Function (mathematics) ,Biochemistry ,Distribution (mathematics) ,Data point ,Approximation error ,Statistical physics ,Anisotropy ,Reduction (mathematics) ,Spectroscopy - Abstract
An accurate description of global tumbling of a protein is essential for correct analysis and interpretation of internal dynamics and thermodynamics. The accurate fitting of global tumbling parameters is affected by the number of experimental relaxation data points available for analysis, the distribution of data points over the domain of the function describing the tumbling, the measurement error associated with the data, the error associated with use of an approximate functional form, and errors in the protein structure. We present an analysis of the influence of these factors on the error in global tumbling parameters and the corresponding error in the calculated T 1/T 2 values. We find that reduction of experimental and approximation error can compensate for a less-than-ideal quantity or distribution of data points, and that accurate parameters can be obtained for proteins with highly anisotropic distributions of bond vectors, as illustrated using the helical bundle protein G-CSF. This indicates that proteins with anisotropic distributions, such as the helical bundle class of proteins, should not summarily be excluded when selecting proteins for dynamic and thermodynamic analyses of 15N backbone relaxation measurements.
- Published
- 2002
24. Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR11Edited by P. E. Wright
- Author
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Theresa Ramelot and Linda K. Nicholson
- Subjects
chemistry.chemical_classification ,education.field_of_study ,biology ,Amyloid beta ,Chemistry ,Population ,Peptide ,Protein structure ,Biochemistry ,Structural Biology ,biology.protein ,Amyloid precursor protein ,Biophysics ,Phosphorylation ,Peptide bond ,education ,Molecular Biology ,Peptide sequence - Abstract
The cytoplasmic tail of the amyloid precursor protein (APPc) interacts with several cellular factors implicated in intracellular signaling or proteolytic production of amyloid beta peptide found in senile plaques of Alzheimer's disease patients. APPc contains two threonine residues (654 and 668 relative to APP695, or 6 and 20 relative to APPc) and a serine residue (655 or 7, respectively) that are known to be phosphorylated in vivo and may play regulatory roles in these events. We show by solution NMR spectroscopy of a 49 residue cytoplasmic tail peptide (APP-C) that in all three cases, phosphorylation induces changes in backbone dihedral angles that can be attributed to formation of local hydrogen bonds between the phosphate group and nearby amide protons. Phosphorylation of S7 also induces chemical shift changes in the hydrophobic cluster (residues I8-V13), indicating additional medium-range effects. The most pronounced changes occur upon phosphorylation of T20, a neuron-specific phosphorylation site, where the N-terminal helix capping box previously characterized for this region is altered. Characterization of torsion angles and transient hydrogen bonds indicates that prolyl isomerization of the pThr-Pro peptide bond results from both destabilization of the N-terminal helix capping box and stabilization of the cis isomer by transient hydrogen bonds. The significant population of the cis isomer (9 %) present after phosphorylation of T20 suggests a potential role of selective recognition of cis versus trans isomers in response to phosphorylation of APP. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the cytoplasmic tail of APP to alter specificity and affinity of binding to cytosolic partners, particularly in response to the abnormal phosphorylation events associated with Alzheimer's disease.
- Published
- 2001
25. [Untitled]
- Author
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Linda K. Nicholson, Chunyu Wang, Shohei Koide, and Norma H. Pawley
- Subjects
Quantitative Biology::Biomolecules ,Chemistry ,Chemical exchange ,Perturbation (astronomy) ,Rotational diffusion ,Improved method ,Biochemistry ,Synthetic data sets ,Bacterial vaccine ,Nuclear magnetic resonance ,Protein structure ,Chemical physics ,Anisotropy ,Spectroscopy - Abstract
Although an accurate description of global tumbling of a protein is essential for correct analysis of internal motions. proper distinction between the effects of anisotropic rotational diffusion and conformational exchange has remained a challenge. We present a novel two-part filtering procedure designed specifically to distinguish between the effects of anisotropy and conformational exchange. The efficacy of this method is assessed using synthetic data sets. The method is then applied to two proteins of dramatically different size and shape, OspA and SH3. The large size and extreme anisotropy of OspA provide a challenging case, where conformational exchange is a small perturbation of the effects of anisotropy on transverse relaxation rates. Conversely, in the chicken c-Src SH3 domain, with its small size and nearly spherical shape, anisotropy is a small perturbation of the effects of conformational exchange on transverse relaxation rates. Accurate extraction of the global tumbling parameters for each protein allows optimal characterization of conformational exchange processes, as well as ps-ns time scale motions.
- Published
- 2001
26. [Untitled]
- Author
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Jun Xi, Linda K. Nicholson, Chunyu Wang, and Tadhg P. Begley
- Subjects
biology ,Sequence alignment ,Biochemistry ,NEDD8 ,Transport protein ,Protein structure ,Ubiquitin ,Structural Biology ,Phylogenetics ,Genetics ,biology.protein ,Protein folding ,Peptide sequence - Abstract
ThiS is a sulfur carrier protein that plays a central role in thiamin biosynthesis in Escherichia coli. Here we report the solution NMR structure of ThiS, the first for this class of sulfur carrier proteins. Although ThiS shares only 14% sequence identity with ubiquitin, it possesses the ubiquitin fold. This structural homology, combined with established functional similarities involving sulfur chemistry, demonstrates that the eukaryotic ubiquitin and the prokaryotic ThiS evolved from a common ancestor. This illustrates how structure determination is essential in establishing evolutionary links between proteins in which structure and function have been conserved through eons of evolution despite loss of sequence identity. The ThiS structure reveals both hydrophobic and electrostatic surface features that are likely determinants for interactions with binding partners. Comparison with surface features of ubiquitin and ubiquitin homologs SUMO-1, RUB-1 and NEDD8 suggest how Nature has utilized this single fold to incorporate similar chemistry into a broad array of highly specific biological processes.
- Published
- 2001
27. Ligand-Induced Strain in Hydrogen Bonds of the c-Src SH3 Domain Detected by NMR
- Author
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Linda K. Nicholson, Chunyu Wang, Stephan Grzesiek, and Florence Cordier
- Subjects
Models, Molecular ,Hydrogen ,Molecular Sequence Data ,Proto-Oncogene Proteins pp60(c-src) ,Low-barrier hydrogen bond ,chemistry.chemical_element ,Ligands ,Protein Structure, Secondary ,SH3 domain ,src Homology Domains ,Protein structure ,Structural Biology ,Animals ,Amino Acid Sequence ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Binding Sites ,Hydrogen bond ,Hydrogen Bonding ,Ligand (biochemistry) ,Crystallography ,chemistry ,Protons ,Chickens ,Macromolecule - Abstract
Changes in the molecular conformation of proteins can result from a variety of perturbations, and can play crucial roles in the regulation of biological activity. A new solution NMR method has been applied to monitor ligand-induced changes in hydrogen bond geometry in the chicken c-Src SH3 domain. The structural response of this domain to ligand binding has been investigated by measuring trans-hydrogen bond (15)N-(13)C' scalar couplings in the free state and when bound to the high affinity class I ligand RLP2, containing residues RALPPLPRY. A comparison between hydrogen bonds in high resolution X-ray structures of this domain and those observed via (h3)J(NC') couplings in solution shows remarkable agreement. Two backbone-to-side-chain hydrogen bonds are observed in solution, and each appears to play a role in stabilization of loop structure. Reproducible ligand-induced changes in trans-hydrogen bond scalar couplings are observed across the domain that translate into changes in hydrogen bond length ranging between 0.02 to 0.12 A. The observed changes can be rationalized by an induced fit mechanism in which hydrogen bonds across the protein participate in a compensatory response to forces imparted at the protein-ligand interface. Upon ligand binding, mutual intercalation of the two Leu-Pro segments of the ligand between three aromatic side-chains protruding from the SH3 surface wedges apart secondary structural elements within the SH3 domain. This disruption is transmitted in a domino-like effect across the domain through networks of hydrogen bonded peptide planes. The unprecedented resolution obtained demonstrates the ability to characterize subtle structural rearrangements within a protein upon perturbation, and represents a new step in the endeavor to understand how hydrogen bonds contribute to the stabilization and function of biological macromolecules.
- Published
- 2000
28. Protein Dynamics Measurements by TROSY-Based NMR Experiments
- Author
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Youlin Xia, Guang Zhu, Kong-Hung Sze, and Linda K. Nicholson
- Subjects
Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Time Factors ,Resolution (mass spectrometry) ,Calmodulin ,Protein Conformation ,Biophysics ,Analytical chemistry ,Amide proton ,Sensitivity and Specificity ,Biochemistry ,Xenopus laevis ,Nuclear magnetic resonance ,Triple-resonance nuclear magnetic resonance spectroscopy ,Animals ,Nitrogen Isotopes ,biology ,Chemistry ,Protein dynamics ,Relaxation (NMR) ,Deuterium ,Condensed Matter Physics ,Amides ,Cold Temperature ,biology.protein ,Heteronuclear single quantum coherence spectroscopy - Abstract
The described TROSY-based experiments for investigating backbone dynamics of proteins make it possible to elucidate internal motions in large proteins via measurements of T(1), T(2), and NOE of backbone (15)N nuclei. In our proposed sequences, the INEPT sequence is eliminated and the PEP sequence is replaced by the ST2-PT sequence from the HSQC-based experiments. This has the benefit of shortening the pulse sequences by 5.4 ms (=1/2J) and results in an increase in the intrinsic sensitivity of the proposed TROSY-based experiments. The TROSY-based experiments are on average of 13% more sensitive than the corresponding HSQC-based experiments on a uniformly (15)N-labeled Xenopus laevis calcium-bound calmodulin sample on a 750-MHz spectrometer at 5 degrees C. The amide proton linewidths of the TROSY-based experiments are 2-13 Hz narrower than those of the HSQC experiments. More sensitivity gain and higher resolution are expected if the protein sample is deuterated.
- Published
- 2000
29. RANTES Binding and Down-Regulation by a Novel Human Herpesvirus-6 β Chemokine Receptor
- Author
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Ursula A. Gompels, C Mattick, Linda K. Nicholson, Richard S. B. Milne, Prema Devaraj, and Antonio Alcami
- Subjects
CCR1 ,Genes, Viral ,Chemokine receptor CCR5 ,Herpesvirus 6, Human ,Molecular Sequence Data ,Immunology ,Down-Regulation ,C-C chemokine receptor type 6 ,Ligands ,Cell Line ,Viral Proteins ,Chemokine receptor ,GTP-Binding Proteins ,Tumor Cells, Cultured ,Animals ,Humans ,Immunology and Allergy ,Amino Acid Sequence ,CCL13 ,Chemokine CCL5 ,Viral Structural Proteins ,biology ,Epithelial Cells ,Molecular biology ,Chemokines, CC ,biology.protein ,Receptors, Virus ,XCL2 ,Receptors, Chemokine ,K562 Cells ,CC chemokine receptors ,Protein Binding ,CCL21 - Abstract
The human herpesvirus 6 (HHV-6) U51 gene defines a new family of betaherpesvirus-specific genes encoding multiple transmembrane glycoproteins with similarity to G protein-coupled receptors, in particular, human chemokine receptors. These are distinct from the HHV-6 U12 and HCMV US28 family. In vitro transcription and translation as well as transient cellular expression of U51 showed properties of a multiple transmembrane protein with a 30-kDa monomer as well as high m.w. aggregates or oligomers. Transient cellularly expressed U51 also appeared to form dimeric intermediates. Despite having only limited sequence similarity to chemokine receptors, U51 stably expressed in cell lines showed specific binding of the CC chemokine RANTES and competitive binding with other β chemokines, such as eotaxin; monocyte chemoattractant protein 1, 3, and 4; as well as the HHV-8 chemokine vMIPII. In epithelial cells already secreting RANTES, U51 expression resulted in specific transcriptional down-regulation. This correlated with reduced secretion of RANTES protein into the culture supernatants. Regulation of RANTES levels may alter selective recruitment of circulating inflammatory cells that the virus can infect and thus could mediate the systemic spread of the virus from initial sites of infection in epithelia. Alternatively, chemokine regulation could modulate a protective inflammatory response to aid the spread of virus by immune evasion. Such mimicry, by viral proteins, of host receptors leading to down-regulation of chemokine expression is a novel immunomodulatory mechanism.
- Published
- 2000
30. Transient Structure of the Amyloid Precursor Protein Cytoplasmic Tail Indicates Preordering of Structure for Binding to Cytosolic Factors
- Author
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Theresa Ramelot, Linda K. Nicholson, and Lisa N. Gentile
- Subjects
Models, Molecular ,Cytoplasm ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Nerve Tissue Proteins ,Peptide ,Biochemistry ,Protein Structure, Secondary ,Turn (biochemistry) ,Amyloid beta-Protein Precursor ,Cytosol ,Protein structure ,Amyloid precursor protein ,Humans ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,Adaptor Proteins, Signal Transducing ,chemistry.chemical_classification ,biology ,Chemistry ,P3 peptide ,Hydrogen Bonding ,Hydrogen-Ion Concentration ,Peptide Fragments ,biology.protein ,Biophysics ,Amyloid precursor protein secretase ,Alpha helix ,Plasmids ,Protein Binding - Abstract
The cytoplasmic tail of the amyloid precursor protein (APP) appears to play two important roles in the cell through participation in intracellular signaling and proteolytic processing of APP. Hence, knowledge of the structure of the 47 residue cytoplasmic tail of APP is important for understanding the molecular interactions involved in normal cell function as well as in the pathogenesis of Alzheimer's disease. Multidimensional solution NMR spectroscopy has been applied to examine the structural features of a 49-residue peptide (APP-C) containing two N-terminal residues (GS) and the APP cytoplasmic tail, over the pH range of 4.2-7.1. Although the peptide does not adopt a stable folded structure, regions of unstable structure exist over the pH range examined and have been characterized by a combination of H(alpha) chemical shifts, NOE analysis, and (3)J(HNH)(alpha) coupling constants and by identification of transient hydrogen bonds between amide protons and titrating carboxylate groups. These studies extend the work of others [Kroenke et al. (1997) Biochemistry 36, 8145-8152] by identifying an additional nascent helix and a hydrophobic cluster within the N-terminal 20 amino acid residues and by further characterizing the TPEE turn as a helix capping box. The transient structure of APP-C provides insight into the importance of preordering of this cytoplasmic tail in governing specificity and affinity for cytosolic binding partners.
- Published
- 2000
31. Backbone Dynamics of Inactive, Active, and Effector-Bound Cdc42Hs from Measurements of15N Relaxation Parameters at Multiple Field Strengths
- Author
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Adrienne P. Loh, Linda K. Nicholson, Robert E. Oswald, and Wei Guo
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Nitrogen Isotopes ,Chemistry ,Effector ,Stereochemistry ,Molecular Sequence Data ,A protein ,Cell Cycle Proteins ,Nuclear magnetic resonance spectroscopy ,Nanosecond ,Biochemistry ,Crystallography ,Cdc42 GTP-Binding Protein ,GTP-Binding Proteins ,Proton NMR ,Amino Acid Sequence ,Protons ,Ras superfamily ,cdc42 GTP-Binding Protein ,Peptide sequence - Abstract
Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs and found that the regions involved in effector (Switch I) and regulator (Switch II) actions are partially disordered [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. Recently, we used a 46-amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes the beta2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the backbone dynamics of three constructs of [(15)N]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using (15)N-(1)H NMR measurements of T(1), T(1)(rho), and the steady-state NOE at three magnetic field strengths. Residue-specific values of the generalized order parameters (S(s)(2) and S(f)(2)), local correlation time (tau(e)), and exchange rate (R(ex)) were obtained using the Lipari-Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) motions on a nanosecond time scale, whereas those in Switch II experience low-amplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhibits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs-GMPPCP and Cdc42Hs-PBD46. Overall, significant flexibility was observed mainly in the regions of Cdc42Hs that are involved in protein-protein interactions (Switch I, Switch II, and Insert), and flexibility was reduced upon interaction with a protein ligand. These results suggest that protein flexibility is important for high-affinity binding interactions.
- Published
- 1999
32. Prolyl cis-trans Isomerization as a Molecular Timer in Crk Signaling
- Author
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Kun Ping Lu and Linda K. Nicholson
- Subjects
Magnetic Resonance Spectroscopy ,Proline ,Protein Conformation ,Stereochemistry ,Cell ,Signal transducing adaptor protein ,Cell Biology ,Peptidylprolyl Isomerase ,Proto-Oncogene Proteins c-crk ,Biology ,Article ,Cis trans isomerization ,src Homology Domains ,Adapter molecule crk ,medicine.anatomical_structure ,Isomerism ,Biochemistry ,medicine ,Thermodynamics ,Molecular Biology ,Isomerization ,Signal Transduction - Abstract
Autoinhibition is being widely used in nature to repress otherwise constitutive protein activities and is typically regulated by extrinsic factors. Here we show that autoinhibition can be controlled by an intrinsic intramolecular switch afforded by prolyl cis-trans isomerization. We find that a proline on the linker tethering the two SH3 domains of the Crk adaptor protein interconverts between the cis and trans conformation. In the cis conformation, the two SH3 domains interact intramolecularly, thereby forming the basis of an autoinhibitory mechanism. Conversely, in the trans conformation Crk exists in an extended, uninhibited conformation that is marginally populated but serves to activate the protein upon ligand binding. Interconversion between the cis and trans, and, hence, of the autoinhibited and activated conformations is accelerated by the action of peptidyl-prolyl isomerases. Proline isomerization appears to make an ideal switch that can regulate the kinetics of activation, thereby modulating the dynamics of signal response.
- Published
- 2007
33. Heteronuclear NMR Studies of the Combined Src Homology Domains 2 and 3 of pp60 c-Src: Effects of Phosphopeptide Binding
- Author
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Geerten W. Vuister, David Shalloway, Linda K. Nicholson, Marco Tessari, Lisa N. Gentile, and Stephen J. Taylor
- Subjects
Models, Molecular ,Phosphopeptides ,Protein Conformation ,Stereochemistry ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Proto-Oncogene Proteins pp60(c-src) ,Peptide binding ,macromolecular substances ,Nuclear Overhauser effect ,SH2 domain ,Sensitivity and Specificity ,Biochemistry ,Protein Structure, Secondary ,SH3 domain ,src Homology Domains ,Nuclear magnetic resonance ,Escherichia coli ,Amino Acid Sequence ,Cloning, Molecular ,Nuclear Magnetic Resonance, Biomolecular ,Glutathione Transferase ,Binding Sites ,Chemistry ,Phosphopeptide ,Chemical shift ,Heteronuclear molecule ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.
- Published
- 1997
34. Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy
- Author
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Peter J. Domaille, Chong Hwan Chang, Andrew P. Hinck, Linda K. Nicholson, Yun Xing Wang, Joshua D. Kaufman, Toshimasa Yamazaki, Stephen J. Stahl, Paul T. Wingfield, Patrick Y.S. Lam, and Dennis A. Torchia
- Subjects
Protease ,biology ,Stereochemistry ,Chemistry ,medicine.medical_treatment ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,NMR spectra database ,Crystallography ,Protein structure ,HIV-1 protease ,Heteronuclear molecule ,biology.protein ,medicine ,Molecule ,HIV Protease Inhibitor ,Molecular Biology - Abstract
The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.
- Published
- 1996
35. Novel Bivalent Interaction between VASP-EVH1 And Zyxin is Critical for Binding Orientation
- Author
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Eric B. Gibbs, Alexander I. Greenwood, Lucila A. Acevedo, Scott A. Showalter, and Linda K. Nicholson
- Subjects
EVH1 domain ,Chemistry ,Ran ,Biophysics ,Phosphorylation ,Nuclear magnetic resonance spectroscopy ,Binding site ,Cytoskeleton ,Actin ,Zyxin - Abstract
The interaction of EVH1 domain of the actin polymerization regulatory protein VASP with the cytoskeletal protein Zyxin shows a novel secondary binding site by NMR spectroscopy. Previous studies had shown the importance of the Y39 phosphorylation in EVH1 for co-localization of VASP and Zyxin in the cell. We found a novel secondary binding site that is located in the structural vicinity of Y39. Phosphomimetic mutation Y39E eliminates binding to this secondary interaction site. By analyzing NMR populations, we determined that the secondary binding site provides an orientation preference for the four-motif Zyxin ligand. We further ran double/triple resonance NMR experiments, including carbon-detected, to determine the resonance assignments and structure of a chimera that includes EVH1 and motifs two-four of Zyxin. This structure shows EVH1 bound simultaneously and specifically to two of the four proline-rich binding motifs in Zyxin. This works reveals a secondary binding site in EVH1 that could represent a previously unrecognized coordination mechanism for partners with multiple proline-rich binding motifs.
- Published
- 2016
36. Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at threonine668
- Author
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Evgenii L. Kovrigin, Linda K. Nicholson, Kun Ping Lu, Monique J. Rogals, Alexander I. Greenwood, and Soumya De
- Subjects
Models, Molecular ,Biochemistry ,Article ,WW domain ,Amyloid beta-Protein Precursor ,Protein structure ,Isomerism ,Protein Interaction Mapping ,Humans ,Binding site ,Phosphorylation ,Nuclear Magnetic Resonance, Biomolecular ,Molecular switch ,Binding Sites ,biology ,Chemistry ,Tryptophan ,Isothermal titration calorimetry ,Peptidylprolyl Isomerase ,Protein Structure, Tertiary ,NIMA-Interacting Peptidylprolyl Isomerase ,Crystallography ,Kinetics ,Biophysics ,biology.protein ,Thermodynamics ,Isomerization ,Cis–trans isomerism ,Binding domain ,Protein Binding - Abstract
Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.
- Published
- 2012
37. Proline Isomer-Specific Antibodies Reveal the Early Pathogenic Tau Conformation in Alzheimer's Disease
- Author
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Xiao Zhen Zhou, Kun Ping Lu, Alexander I. Greenwood, Lester I. Binder, Eileen H. Bigio, Linda K. Nicholson, Kazuhiro Nakamura, and Sarah Denial
- Subjects
Cell signaling ,Proline ,tau Proteins ,Biology ,Hippocampus ,General Biochemistry, Genetics and Molecular Biology ,Article ,Dephosphorylation ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Isomerism ,Alzheimer Disease ,medicine ,Animals ,Humans ,NIMA-Interacting Peptidylprolyl Isomerase ,030304 developmental biology ,Aged ,Peptidylprolyl isomerase ,Cerebral Cortex ,0303 health sciences ,Biochemistry, Genetics and Molecular Biology(all) ,Kinase ,Peptidylprolyl Isomerase ,medicine.disease ,3. Good health ,Frontal Lobe ,Mice, Inbred C57BL ,Disease Models, Animal ,Biochemistry ,PIN1 ,Phosphorylation ,Alzheimer's disease ,030217 neurology & neurosurgery - Abstract
Summarycis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.
- Published
- 2012
38. Structural analysis of highly oriented poly(p-phenylene-terephthalamide) by 15N solid-state nuclear magnetic resonance
- Author
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Teruaki Fujito, Linda K. Nicholson, Makoto Demura, Timothy A. Cross, Tetsuo Asakura, Mamoru Imanari, and Joo-Hong Yeo
- Subjects
Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Radiation ,Materials science ,Molecular Structure ,Nitrogen Isotopes ,Polymers ,Phthalic Acids ,General Chemistry ,Aramid ,Crystallography ,Solid-state nuclear magnetic resonance ,Poly(p-phenylene) ,Perpendicular ,Molecule ,Fiber ,Spectroscopy ,Instrumentation ,Principal axis theorem - Abstract
The structure of an aromatic polyamide, poly(p-phenylene-terephthalamide) (PPTA), was studied in the solid state using 15N nuclear magnetic resonance (NMR) spectroscopy. Spectra of uniaxially aligned molecules placed with the axis of alignment both parallel with and perpendicular to the applied magnetic field were analyzed to yield the orientations of specific molecular bonds with respect to the fiber axis. The 15N chemical shift tensor was characterized by simulating powder pattern spectra of both PPTA and a model compound, benzanilide. Chemical shift and dipolar coupled chemical shift line shapes were calculated through Euler angle transformations from the principal axis system (PAS) reference frame to the fiber axis system (FAS) frame. The orientations of NH and NC' bonds in PPTA are determined as well as the orientational distribution of the PPTA fiber axis. The structural parameters determined for PPTA are compared with those obtained by X-ray diffraction.
- Published
- 1994
39. Secondary structure and signal assignments of human-immunodeficiency-virus-1 protease complexed to a novel, structure-based inhibitor
- Author
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Peter J. Domaille, Toshimasa Yamazaki, Dennis A. Torchia, Paul T. Wingfield, Sharon Campbell-Burk, Linda K. Nicholson, Joshua D. Kaufman, and Stephan J. Stahl
- Subjects
Magnetic Resonance Spectroscopy ,Stereochemistry ,medicine.medical_treatment ,Molecular Sequence Data ,Plasma protein binding ,Biology ,Biochemistry ,Protein Structure, Secondary ,Protein structure ,HIV Protease ,Escherichia coli ,medicine ,Point Mutation ,Urea ,Amino Acid Sequence ,Cloning, Molecular ,Protein secondary structure ,Peptide sequence ,chemistry.chemical_classification ,Protease ,Mutagenesis ,Azepines ,HIV Protease Inhibitors ,Recombinant Proteins ,Solvent ,Enzyme ,chemistry ,Mutagenesis, Site-Directed ,Protein Binding - Abstract
We report comprehensive NMR studies in solution of the human-immunodeficiency-virus (HIV)-1 protease. Stable solutions of the protease were obtained by complexing the protein to a designed cyclic urea inhibitor DMP 323. A variety of triple-resonance experiments provided essentially complete 1H, 13C and 15N NMR signal assignments of the protease. These assignments, together with short-range NOE constraints, coupling constants and hydrogen-exchange data, yielded the secondary structure of the protease in solution. The results reported herein open the way to the determination of the high-resolution three-dimensional solution structures of protease/inhibitor complexes, as well as to studies of protease dynamics and solvent interactions.
- Published
- 1994
40. Complete Determination of the Pin1 Catalytic Domain Thermodynamic Cycle by NMR Lineshape Analysis
- Author
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Monique J. Rogals, Alexander I. Greenwood, Kun Ping Lu, Evgenii L. Kovrigin, Soumya De, and Linda K. Nicholson
- Subjects
Peptidylprolyl isomerase ,Isomerase activity ,Binding Sites ,Stereochemistry ,Chemistry ,Context (language use) ,Isomerase ,Peptidylprolyl Isomerase ,Biochemistry ,Article ,Enzyme catalysis ,NIMA-Interacting Peptidylprolyl Isomerase ,Kinetics ,Catalytic Domain ,Biocatalysis ,Thermodynamics ,Phosphorylation ,Isomerization ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Cis–trans isomerism ,Heteronuclear single quantum coherence spectroscopy - Abstract
The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, $$ k_{cat}^{cis} $$ and apparent Michaelis constants, $$ K_{M}^{App} $$ . By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13C–1H constant time HSQC spectra to determine $$ k_{cat}^{cis} $$ , $$ k_{cat}^{trans} $$ , $$ K_{D}^{cis} $$ , and $$ K_{D}^{trans} $$ for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.
- Published
- 2011
41. Bicelle-Bound Solid-State NMR Structural Studies and Membrane-Permeabilizing Activities of Piscidin 1 and Piscidin 3: Implications for Mode of Antimicrobial Action
- Author
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Anna A. De Angelis, Daryl M. Berke, Linda K. Nicholson, Matthew K. Baxter, Jason A. McGavin, Nedzada Smajic, Myriam Cotten, Jolita Seckute, Stanley J. Opella, Caitlin M. Burzynski, and Nina B. Kraus
- Subjects
chemistry.chemical_classification ,Crystallography ,Membrane ,chemistry ,Solid-state nuclear magnetic resonance ,Antimicrobial peptides ,Amphiphile ,Biophysics ,Peptide ,Model lipid bilayer ,Lipid bilayer ,Micelle - Abstract
Bicelles represent a novel preparation of hydrated lipid bilayers, which can be used to study membrane-associated proteins under physiologically-relevant conditions. Large bicelles can be oriented within a magnetic field, enabling the determination of high-resolution peptide structures and angles of insertion within a lipid membrane via solid-state Nuclear Magnetic Resonance (NMR).Piscidin, an amphipathic, antimicrobial peptide found in hybrid striped bass, plays a major role in host defense. It is effective against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus and HIV-1. The peptide is known to have an alpha-helical conformation when bound to anionic lipid membranes that mimic the surface of bacterial membranes.We have investigated the use of bicelles in the study of piscidin. 15N NMR spectra show that piscidin has been successfully aligned in magnetically oriented bicelles. 31P NMR studies, which show that piscidin disrupts bicelle-forming lipids, have helped us better understand its mode of action. To complement these backbone solid-state NMR studies of piscidin, we have used fluorescent-dye leakage experiments with various phospholipids and have performed solution NMR to determine the charge state of the histidine side chains in the presence of micelles and investigate their possible role in mediating important peptide-lipid interactions. The long term goal of this project is to improve our understanding of structure function relationships in an interesting family of antimicrobial peptides. This knowledge could be used to design potent antimicrobial pharmaceuticals that minimize bacterial resistance.
- Published
- 2011
- Full Text
- View/download PDF
42. Structural analysis of uniaxially aligned polymers using solid-state nitrogen-15 NMR
- Author
-
Joo Hong Yeo, Makoto Demura, Teruaki Fujito, Mamoru Imanari, Linda K. Nicholson, Timothy A. Cross, Takuro Itoh, and Tetsuo Asakura
- Subjects
Inorganic Chemistry ,chemistry.chemical_classification ,Materials science ,Polymers and Plastics ,chemistry ,Chemical engineering ,Organic Chemistry ,Polymer chemistry ,Materials Chemistry ,Solid-state ,chemistry.chemical_element ,Polymer ,Nitrogen - Published
- 1993
43. A method for studying the structure of uniaxially aligned biopolymers using solid state15N-nmr: Application toBombyx mori silk fibroin fibers
- Author
-
Linda K. Nicholson, Makoto Demura, Timothy A. Cross, and Tetsuo Asakura
- Subjects
Diffraction ,Magnetic Resonance Spectroscopy ,Nitrogen Isotopes ,Protein Conformation ,Chemistry ,Organic Chemistry ,Silk ,Biophysics ,Proteins ,Fibroin ,General Medicine ,Bombyx ,Biochemistry ,Biomaterials ,NMR spectra database ,Nuclear magnetic resonance ,Solid-state nuclear magnetic resonance ,Chemical physics ,Animals ,Insect Proteins ,Molecule ,Fibroins ,Anisotropy ,Spectroscopy ,Fiber diffraction - Abstract
Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction. In the present work, a new method is developed in which solid state 15N-nmr spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield the orientations of specific molecular bonds. Analytical expressions are derived that utilize spectral features read from 15N chemical shift anisotropy line shapes to calculate a discrete number of possible orientations for a specific site. The 15N-1H dipolar interaction is employed to further narrow the number of unique orientations possible for a given site. With this method, a neighborhood of possible orientations is quickly determined, and full line-shape simulations within this region of allowed space can be performed to refine the limits of orientation. This technique demonstrates the use of a single type of isotopic label to determine the orientation of a specific molecular group such as a peptide plane within a protein. Results from the application of this method to the Bombyx mori silk fibroin protein provide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies. © 1993 John Wiley & Sons, Inc.
- Published
- 1993
44. Determining the Charge State of Histidine Side Chains in Antimicrobial Piscidin By Nuclear Magnetic Resonance
- Author
-
Matthew K. Baxter, Jolita Seckute, Myriam Cotten, Jason A. McGavin, and Linda K. Nicholson
- Subjects
chemistry.chemical_compound ,Nuclear magnetic resonance ,Biochemistry ,Chemistry ,Antimicrobial peptides ,Side chain ,Biophysics ,Context (language use) ,Sodium dodecyl sulfate ,Antibacterial activity ,Antimicrobial ,Micelle ,Histidine - Abstract
Piscidins constitute a family of three antimicrobial peptides discovered in the mast cells of hybrid striped bass. These peptides, which are highly cationic, contain several arginine and histidine residues. While piscidin 1 is the most antimicrobial and hemolytic isoform, piscidin 3, which has slightly lower antimicrobial activity, is significantly less hemolytic. One of the most striking differences between piscidin 1 and 3 is the substitution of glycine for the histidine at position 17 in piscidin 1.As part of its mechanism of action, piscidin recognizes negatively charged microbial membranes. Therefore, studying the interactions of the piscidin with lipids can help us better understand the chemical basis of its antimicrobial and hemolytic effects. Because physiological pH is around 7.4, and the average pKa of histidine side chains is around 6.0, a detailed study of the histidine side chains in piscidin 1 and 3 is needed to discern the charge state of the peptides under physiological conditions. In this research, we used solution nuclear magnetic resonance to obtain the pKa of the histidine side chains of piscidin bound to sodium dodecyl sulfate micelles. Heteronuclear multiple quantum coherence experiments were performed on piscidin 1 and 3 containing 15N-side chain labeled histidines. 15N and 1H chemical shifts were recorded as a function of pH to determine the titration curve of each histidine residue. The results will be discussed in the context of structure-function relationships in membrane-active peptides. The knowledge gained from these studies can help identify common principles that will facilitate the design of pharmaceuticals with broad-spectrum antibacterial activity, minimum induction of bacterial resistance, and low toxicity to mammalian cells.
- Published
- 2010
- Full Text
- View/download PDF
45. The measurement of heteronuclear transverse relaxation times in ax3 spin systems via polarization-transfer techniques
- Author
-
Christian Griesinger, Lewis E. Kay, T.E Bull, Linda K. Nicholson, Harald Schwalbe, Dennis A. Torchia, and Ad Bax
- Subjects
Spin–spin relaxation ,Magnetization ,Heteronuclear molecule ,Condensed matter physics ,Spins ,Chemistry ,Relaxation (NMR) ,General Engineering ,Pulse sequence ,Nuclear magnetic resonance spectroscopy ,Polarization (waves) ,Molecular physics - Abstract
Pulse schemes for the measurement of 13C transverse relaxation times in AX33 spin systems are described which make use of the sensitive 1H spin for detection. The experiments are based on reverse-DEPT and reverse-INEPT polarization-transfer sequences. It is shown that relaxation rates obtained from 13C-direct-observe and from polarization-transfer experiments are identical only if magnetization from each of the 13C transitions is transferred equally to the detected 1H spins. This requires judicious choices in pulse angles and delays in reverse-DEPT and reverse-INEPT experiments. For application to macromolecules, experimental and theoretical results suggest that polarization-transfer schemes based on reverse INEPT are superior to reverse-DEPT-based sequences and give results which are in good agreement with values measured via 13C-observe methods.
- Published
- 1992
46. Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins
- Author
-
Dennis A. Torchia, Frank Delaglio, Lewis E. Kay, Linda K. Nicholson, and Adriaan Bax
- Subjects
Spin–spin relaxation ,Nuclear magnetic resonance ,Heteronuclear molecule ,Pulse (signal processing) ,Chemistry ,Relaxation (NMR) ,General Engineering ,Spin–lattice relaxation ,Field strength ,Pulse sequence ,Anisotropy - Abstract
The effects of cross correlation between dipolar and chemical-shift anisotropy relaxation interactions on the measurement of heteroatom T1 and T2 relaxation times in proteins is considered. It is shown that such effects can produce errors of approximately 25% in the measurement of 15N transverse relaxation times at a field strength of 11.8 T. Cross correlation has a less significant effect on the measurement of 15N spin-lattice relaxation rates and for proteins the errors in T1 decrease as a function of increasing molecular weight. Nevertheless, for T1 measurements at 11.8 T errors of approximately 15 and 5% are calculated for proteins with correlation times, τc, of 5 and 9 ns, respectively. Pulse sequences which eliminate dipolar and chemical-shift anisotropy cross-correlation effects are described. These sequences are used to make more accurate measurements of 15N T1 and T2 values of staphylococcal nuclease and to determine errors in these parameters that result when cross correlations are present.
- Published
- 1992
47. A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB
- Author
-
Yi-Pin Lin, Sean P. McDonough, Alexander I. Greenwood, Linda K. Nicholson, Yogendra Sharma, Yung-Fu Chang, and Weiwei Yan
- Subjects
Amino Acid Motifs ,Molecular Sequence Data ,Biophysics ,Cell Culture Techniques ,Biochemistry ,Article ,Bacterial Adhesion ,Dogs ,Leptospira ,Animals ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Antigens, Bacterial ,biology ,C-terminus ,Cell Biology ,biology.organism_classification ,Molecular biology ,Fibronectins ,Protein Structure, Tertiary ,Bacterial adhesin ,Fibronectin ,biology.protein ,Leptospira interrogans ,Binding domain - Abstract
Infection by pathogenic strains of Leptospira hinges on the pathogen's ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708-1712 containing sequence LIPAD with a beta-strand and nascent helical structure. This motif binds to 15th type III modules (15F(3)) (K(D)=10.70 microM), and association (k(on)=600 M(-1)s(-1)) and dissociation (k(off)=0.0129 s(-1)) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB(1706-1716) blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB(1706-1716) in cellular adhesion. These data indicate that the LIPAD residues (LigB(1708-1712)) of the Leptospira interrogans LigB protein bind 15F(3) of Fn at a novel binding site, and this interaction contributes to adhesion to host cells.
- Published
- 2009
48. Fibronectin binds to and induces conformational change in a disordered region of leptospiral immunoglobulin-like protein B
- Author
-
Yung-Fu Chang, Alexander I. Greenwood, Yi-Pin Lin, Linda K. Nicholson, Sean P. McDonough, and Yogendra Sharma
- Subjects
Circular dichroism ,Conformational change ,Antigens, Bacterial ,Isothermal titration calorimetry ,Cell Biology ,Plasma protein binding ,Biology ,Biochemistry ,Molecular biology ,Fibronectins ,Protein Structure, Tertiary ,chemistry.chemical_compound ,Kinetics ,chemistry ,Protein Structure and Folding ,Leptospirosis ,Binding site ,Leptospira interrogans ,Guanidine ,Molecular Biology ,Protein secondary structure ,Heteronuclear single quantum coherence spectroscopy ,Protein Binding - Abstract
Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (K(D) = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (K(av)) measured with gel permeation chromatography, a high hydrodynamic radius (R(h)) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional (15)N-(1)H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a beta-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a beta-strand-rich structure, suggestive of the known beta-zipper mode of NTD binding.
- Published
- 2009
49. Elucidation of a pH-folding switch in the Pseudomonas syringae effector protein AvrPto
- Author
-
Soumya De, Linda K. Nicholson, Jolita Seckute, Aaron Oswald, Samuel A. Schueler, and Jennifer E. Dawson
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Folding ,Multidisciplinary ,Effector ,Temperature ,Titrimetry ,Pseudomonas syringae ,Biology ,Hydrogen-Ion Concentration ,Biological Sciences ,Bacterial effector protein ,Transport protein ,Cell biology ,Type three secretion system ,Protein Structure, Tertiary ,Bacterial Proteins ,Cytoplasm ,Protein folding ,Secretion ,Acids ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly conserved type III secretion system (T3SS) provides a regulated conduit between the bacterial and host cytoplasm for delivery of a specific set of bacterial effector proteins that serve to disrupt host signaling and metabolism for the benefit of the bacterium. Remarkably, the inner diameter of the T3SS apparatus requires that effector proteins pass through in at least a partially unfolded form. AvrPto, an effector protein of the plant pathogen Pseudomonas syringae , adopts a helical bundle fold of low stability (Δ G F→U = 2 kcal/mol at pH 7, 26.6 °C) and offers a model system for chaperone-independent secretion. P. syringae effector proteins encounter a pH gradient as they translocate from the bacterial cytoplasm (mildly acidic) into the host cell (neutral). Here, we demonstrate that AvrPto possesses a pH-sensitive folding switch controlled by conserved residue H87 that operates precisely in the pH range expected between the bacterial and host cytoplasm environments. These results provide a mechanism for how a bacterial effector protein employs an intrinsic pH sensor to unfold for translocation via the T3SS and refold once in the host cytoplasm and provide fundamental insights for developing strategies for delivery of engineered therapeutic proteins to target tissues.
- Published
- 2009
50. Solid-state nuclear magnetic resonance derived model for dynamics in the polypeptide backbone of the gramicidin a channel
- Author
-
Timothy A. Cross, Linda K. Nicholson, and Quincy Teng
- Subjects
Magnetic Resonance Spectroscopy ,Viscosity ,Bilayer ,Lipid Bilayers ,Molecular Sequence Data ,Gramicidin ,Temperature ,Analytical chemistry ,Molecular physics ,Ion Channels ,Spectral line ,Magnetic field ,chemistry.chemical_compound ,Molecular dynamics ,Amplitude ,Models, Chemical ,Solid-state nuclear magnetic resonance ,chemistry ,Structural Biology ,Phase (matter) ,Amino Acid Sequence ,Molecular Biology - Abstract
The dynamics of the backbone of the gramicidin A transmembrane cation channel in dimyristoylphosphatidylcholine bilayers have been investigated using solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopy. With the temperature-dependent fluidity of the bilayer, the rates of motions in the helical gramicidin channel can be modulated. It is shown that in the gel phase, all substantial motions of the channel are slow on the timescale of the n.m.r. experiment (3.5 kHz). The use of oriented samples in which the axis of global channel rotation is aligned parallel to the magnetic field enables separation of global and local dynamics. Spectra obtained from oriented bilayer samples containing single-site 15N-labeled gramicidin at 8 degrees C are analyzed to yield a spatial model for local backbone motion. This model includes the axis of motion, the mean orientation, and the maximum amplitude of displacement for individual peptide planes. Specific sites in the first turn of the amino terminus were investigated, with emphasis on the Ala3 and Leu4 linkages, for which the orientation of the 15N chemical shift tensor with respect to the molecular frame has been determined. The effect of two well-characterized bilayer defect structures, parabolic focal conics and oily streaks, is included in the spectral simulations. It is found that only relatively small amplitude motions are possible at the two sites, with amplitudes of not more than +/- 8 degrees and +/- 15 degrees for the Ala3 and Leu4 sites, respectively. Detailed characterization of the bilayer surface geometry in the oriented samples is presently the major limiting factor in the use of this technique for probing the spatial extent of local motions in integral membrane proteins.
- Published
- 1991
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