105 results on '"Ranajeet Ghose"'
Search Results
2. A Conserved Structural Role for the Walker-A Lysine in P-Loop Containing Kinases
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Fatlum Hajredini and Ranajeet Ghose
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P-loop ,protein kinases ,shikimate kinase ,molecular dynamics (MD) ,bacterial tyrosine kinase ,Biology (General) ,QH301-705.5 - Abstract
Bacterial tyrosine kinases (BY-kinases) and shikimate kinases (SKs) comprise two structurally divergent P-loop containing enzyme families that share similar catalytic site geometries, most notably with respect to their Walker-A, Walker-B, and DxD motifs. We had previously demonstrated that in BY-kinases, a specific interaction between the Walker-A and Walker-B motifs, driven by the conserved “catalytic” lysine housed on the former, leads to a conformation that is unable to efficiently coordinate Mg2+•ATP and is therefore incapable of chemistry. Here, using enhanced sampling molecular dynamics simulations, we demonstrate that structurally similar interactions between the Walker-A and Walker-B motifs, also mediated by the catalytic lysine, stabilize a state in SKs that deviates significantly from one that is necessary for the optimal coordination of Mg2+•ATP. This structural role of the Walker-A lysine is a general feature in SKs and is found to be present in members that encode a Walker-B sequence characteristic of the family (Coxiella burnetii SK), and in those that do not (Mycobacterium tuberculosis SK). Thus, the structural role of the Walker-A lysine in stabilizing an inactive state, distinct from its catalytic function, is conserved between two distantly related P-loop containing kinase families, the SKs and the BY-kinases. The universal conservation of this element, and of the key characteristics of its associated interaction partners within the Walker motifs of P-loop containing enzymes, suggests that this structural role of the Walker-A lysine is perhaps a widely deployed regulatory mechanism within this ancient family.
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- 2021
- Full Text
- View/download PDF
3. A model of a MAPK•substrate complex in an active conformation: a computational and experimental approach.
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Sunbae Lee, Mangalika Warthaka, Chunli Yan, Tamer S Kaoud, Andrea Piserchio, Ranajeet Ghose, Pengyu Ren, and Kevin N Dalby
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Medicine ,Science - Abstract
The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)(2-3)-X(2-6)-Φ(A)-X-Φ(B), where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus (7)RK-X(2)-Φ(A)-X-Φ(B) (13). This results in a 2-fold increase in k(cat)/K(m) for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k(cat)/K(m) by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k(cat)/K(m), with little apparent change in k(cat). A peptide that binds to the DRS of ERK2 affects K(m), but not k(cat). Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.
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- 2011
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- View/download PDF
4. ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
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Andrea Piserchio, Kimberly J. Long, Luke S. Browning, Amanda L. Bohanon, Eta A. Isiorho, Kevin N. Dalby, and Ranajeet Ghose
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Multidisciplinary - Abstract
Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α–kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.
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- 2023
5. Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor
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Andrea Piserchio, Eta A. Isiorho, Kevin N. Dalby, and Ranajeet Ghose
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Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2023
6. Structural dynamics of the complex of calmodulin with a minimal functional construct of eukaryotic elongation factor 2 kinase and the role of Thr348 autophosphorylation
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Ranajeet Ghose, Kwangwoon Lee, Andrea Piserchio, Eric A. Kumar, Rinat R. Abzalimov, Kevin N. Dalby, and Kimberly Long
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Elongation Factor 2 Kinase ,Calmodulin ,Full‐Length Papers ,Allosteric regulation ,Mutation, Missense ,Regulatory site ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,Humans ,Phosphorylation ,Protein Structure, Quaternary ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,030302 biochemistry & molecular biology ,Autophosphorylation ,Elongation factor ,Amino Acid Substitution ,biology.protein ,Biophysics ,Translational elongation ,Alpha helix - Abstract
The calmodulin (CaM) activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2 K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF-2 K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution-state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca2+ -CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C-lobe surface, complementary to the one involved in recognizing the calmodulin-binding domain (CBD) of TR, provides a secondary TR-interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca2+ -CaM•TR complex. Solution NMR data suggest that CaMH107K , which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaMH107K to induce TR autophosphorylation compared to wild-type CaM. This article is protected by copyright. All rights reserved.
- Published
- 2021
7. Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase
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Andrea Piserchio, Eta A. Isiorho, Kimberly Long, Amanda L. Bohanon, Eric A. Kumar, Nathan Will, David Jeruzalmi, Kevin N. Dalby, and Ranajeet Ghose
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Multidisciplinary - Abstract
Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348. Biochemical studies predict a calmodulin-mediated activation mechanism for eEF-2K distinct from other calmodulin-dependent kinases. Here, we resolve the atomic details of this mechanism through a 2.3-Å crystal structure of the heterodimeric complex of calmodulin and the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights an intimate association of the kinase with the calmodulin C-lobe, creating an “activation spine” that connects its amino-terminal calmodulin-targeting motif to its active site through a conserved regulatory element.
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- 2022
8. Conserved Link between Catalytic Site Interactions and Global Conformation in P-loop Enzymes
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Ranajeet Ghose and Fatlum Hajredini
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P-loop enzymes, ubiquitous in all of life’s domains and viruses, comprise a monophyletic group with pre-LUCA origins that have differentiated into several three-layered α/β/α− sandwich domain families utilizing a basic β− loop−α−β structural module housing conserved nucleotide-binding Walker-A and Walker-B sequences. We have analyzed a large dataset of P-loop enzyme structures representing both their KG and ASCE branches as proxies for their sampled conformational landscapes. We developed a novel framework to correlate global conformations and local catalytic site geometry, specifically involving the Walker motifs, to identify conserved signatures despite substantial structural and functional diversity. Our results suggest that P-loop enzymes populate global states broadly classifiable as open or closed. In the closed states, that share similar overall geometries irrespective of family, key catalytic site residues are aligned to optimally engage the critical Mg2+ ion suggesting compatibility with the chemical step. These catalytic site interactions are disrupted in the open states resulting in the loss of the Mg2+- coordinating ability yielding conformations incapable of chemistry. In contrast to the closed states, open states are highly diverse, and this variability is facilitated by differential coupling of specific residues that are part of, or spatially proximal to, the Walker motifs with the clade-specific tertiary fold. We suggest that an essential feature in the activation and nucleotide exchange processes for all P-loop enzymes is the universal coupling between global closure and local reorganization of the catalytic site for efficient coordination of Mg2+ that carries a tightly associated cargo, the substrate NTP.
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- 2022
9. Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb
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Sébastien Alphonse, Imane Djemil, Andrea Piserchio, and Ranajeet Ghose
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Multidisciplinary ,Escherichia coli Proteins ,Escherichia coli ,Phosphoprotein Phosphatases ,Membrane Proteins ,Tyrosine ,Phosphorylation ,Protein Tyrosine Phosphatases ,Protein-Tyrosine Kinases - Abstract
Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low–molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structure presents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.
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- 2022
10. Structural Basis for the Calmodulin-Mediated Activation of eEF-2K
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Andrea Piserchio, Eta A. Isiorho, Kimberly Long, Amanda L. Bohanon, Eric A. Kumar, Nathan Will, David Jeruzalmi, Kevin N. Dalby, and Ranajeet Ghose
- Abstract
Translation is a highly energy consumptive process1 tightly regulated for optimal protein quality2 and adaptation to energy and nutrient availability. A key facilitator of this process is the α-kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis3,4. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process5 mechanistically distinct from other calmodulin-dependent kinases6. Here we resolve the atomic details of this mechanism through a 2.3 Å crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a “spine” that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.
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- 2022
11. BY-kinases: Protein tyrosine kinases like no other
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Fatlum Hajredini, Sébastien Alphonse, and Ranajeet Ghose
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Cell Biology ,Molecular Biology ,Biochemistry - Abstract
BY-kinases (for bacterial tyrosine kinases) constitute a family of protein tyrosine kinases that are highly conserved in the bacterial kingdom and occur most commonly as essential components of multicomponent assemblies responsible for the biosynthesis, polymerization, and export of complex polysaccharides involved in biofilm or capsule formation. BY-kinase function has been attributed to a cyclic process involving formation of an oligomeric species, its disassembly into constituent monomers, and subsequent reassembly, depending on the overall phosphorylation level of a C-terminal cluster of tyrosine residues. However, the relationship of this process to the active/inactive states of the enzyme and the mechanism of its integration into the polysaccharide production machinery remain unclear. Here, we synthesize the substantial body of biochemical, cell-biological, structural, and computational data, acquired over the nearly 3 decades since the discovery of BY-kinases, to suggest means by which they fulfill their physiological function. We propose a mechanism involving temporal coordination of the assembly/disassembly process with the autokinase activity of the enzyme and its ability to be dephosphorylated by its counteracting phosphatase. We speculate that this temporal control enables BY-kinases to function as molecular timers that coordinate the diverse processes involved in the synthesis, polymerization, and export of complex sugar derivatives. We suggest that BY-kinases, which deploy distinctive catalytic domains resembling P-loop nucleoside triphosphatases, have uniquely adapted this ancient fold to drive functional processes through exquisite spatiotemporal control over protein-protein interactions and conformational changes. It is our hope that the hypotheses proposed here will facilitate future experiments targeting these unique protein kinases.
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- 2023
12. Abstract 1978: Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase
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Kimberly Long, Andrea Piserchio, Eta Isiorho, Amanda Bohanon, Luke Browning, Ranajeet Ghose, and Kevin Dalby
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Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2023
13. The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase
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Ranajeet Ghose, Kwangwoon Lee, Eric A. Kumar, and Kevin N. Dalby
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Elongation Factor 2 Kinase ,0303 health sciences ,animal structures ,Calmodulin ,biology ,Chemistry ,Kinase ,Full‐Length Papers ,030302 biochemistry & molecular biology ,Context (language use) ,Biochemistry ,Elongation factor ,03 medical and health sciences ,Translational regulation ,Protein biosynthesis ,Biophysics ,biology.protein ,Phosphorylation ,Calcium ,Translational elongation ,Molecular Biology ,030304 developmental biology - Abstract
Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca(2+) thereon. We find that in the absence of Ca(2+), CaM exclusively utilizes its C‐terminal lobe (CaM(C)) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca(2+)‐loaded N‐terminal lobe of CaM (CaM(N)) at increased Ca(2+) levels serves to enhance the affinity further. These latter interactions under Ca(2+) saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca(2+)‐free state. Our data are consistent with a scenario in which Ca(2+) enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.
- Published
- 2019
14. Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo
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Kevin N. Dalby, Matthew Harger, Kenneth Y. Tsai, Pengyu Ren, Rachel M. Sammons, Tamer S. Kaoud, Clint D.J. Tavares, Jacey R. Pridgen, Ramakrishna Edupuganti, Mohamed F. Radwan, Diana Zamora-Olivares, Nancy D. Ebelt, Sabrina X. Van Ravenstein, Eric V. Anslyn, Mangalika Warthaka, Jihyun Park, Micael Cano, William H. Johnson, Ranajeet Ghose, and Andrea Piserchio
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0301 basic medicine ,MAPK/ERK pathway ,Cell cycle checkpoint ,General Physics and Astronomy ,Apoptosis ,medicine.disease_cause ,Dioxanes ,0302 clinical medicine ,lcsh:Science ,Melanoma ,Mitogen-Activated Protein Kinase 1 ,Mutation ,Multidisciplinary ,Molecular medicine ,biology ,Chemistry ,MEK inhibitor ,Small molecule ,3. Good health ,Cell biology ,030220 oncology & carcinogenesis ,Protein Binding ,MAP Kinase Signaling System ,Science ,Mice, Nude ,Kinases ,Drug development ,Mechanism of action ,Molecular Dynamics Simulation ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Cysteine ,Protein Kinase Inhibitors ,Binding Sites ,HEK 293 cells ,Active site ,General Chemistry ,Xenograft Model Antitumor Assays ,Thiazoles ,HEK293 Cells ,030104 developmental biology ,biology.protein ,lcsh:Q - Abstract
Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers., The ERK signalling pathway is activated in many cancers, however ERK1 and ERK2 are difficult to target pharmacologically. Here, the authors identify a small molecule inhibitor that binds covalently to the D-recruitment site of ERK and induces cell death and reduces tumour growth in mice.
- Published
- 2019
15. Comment on mr-2021-54
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Ranajeet Ghose
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- 2021
16. Solution Structure of the Carboxy-Terminal Tandem Repeat Domain of Eukaryotic Elongation Factor 2 Kinase and Its Role in Substrate Recognition
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Fatlum Hajredini, Andrea Piserchio, David H. Giles, Nathan Will, Kevin N. Dalby, and Ranajeet Ghose
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Elongation Factor 2 Kinase ,Models, Molecular ,Protein Conformation, alpha-Helical ,Protein Conformation ,Ribosome ,Article ,Substrate Specificity ,03 medical and health sciences ,0302 clinical medicine ,Peptide Elongation Factor 2 ,Protein Domains ,Tandem repeat ,Structural Biology ,Humans ,Amino Acid Sequence ,Phosphorylation ,Binding site ,Protein kinase A ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Chemistry ,Translation (biology) ,Elongation factor ,Biophysics ,Translational elongation ,030217 neurology & neurosurgery - Abstract
Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K(562–725) that encodes two α-helical repeats. The structure of eEF-2K(562–725) shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Further, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K(562–725). We find that eEF-2K(562–725) engages eEF-2 with an affinity comparable to that of the full-length enzyme. Further, eEF-2K(562–725) is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K(562–725) encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.
- Published
- 2019
17. Targeting ERK beyond the boundaries of the kinase active site in melanoma
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Ranajeet Ghose, Kevin N. Dalby, Rachel M. Sammons, and Kenneth Y. Tsai
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Proto-Oncogene Proteins B-raf ,0301 basic medicine ,MAPK/ERK pathway ,Cancer Research ,MAP Kinase Signaling System ,Mutant ,Druggability ,Biology ,Cell fate determination ,Article ,Protein–protein interaction ,03 medical and health sciences ,0302 clinical medicine ,Catalytic Domain ,medicine ,Extracellular ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Melanoma ,Protein Kinase Inhibitors ,Molecular Biology ,Kinase ,medicine.disease ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer research ,Signal Transduction - Abstract
Extracellular signal-regulated kinase 1/2 (ERK1/2) constitute a point of convergence for complex signaling events that regulate essential cellular processes, including proliferation and survival. As such, dysregulation of the ERK signaling pathway is prevalent in many cancers. In the case of BRAF-V600E mutant melanoma, ERK inhibition has emerged as a viable clinical approach to abrogate signaling through the ERK pathway, even in cases where MEK and Raf inhibitor treatments fail to induce tumor regression due to resistance mechanisms. Several ERK inhibitors that target the active site of ERK have reached clinical trials, however, many critical ERK interactions occur at other potentially druggable sites on the protein. Here we discuss the role of ERK signaling in cell fate, in driving melanoma, and in resistance mechanisms to current BRAF-V600E melanoma treatments. We explore targeting ERK via a distinct site of protein-protein interaction, known as the D-recruitment site (DRS), as an alternative or supplementary mode of ERK pathway inhibition in BRAF-V600E melanoma. Targeting the DRS with inhibitors in melanoma has the potential to not only disrupt the catalytic apparatus of ERK but also its noncatalytic functions, which have significant impacts on spatiotemporal signaling dynamics and cell fate.
- Published
- 2019
18. A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation
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Tamer S. Kaoud, Diana Zamora-Olivares, Vsevolod V. Gurevich, Marc A. Giulianotti, Kevin N. Dalby, Ranajeet Ghose, Richard A. Houghten, Rachel M. Sammons, Ginamarie Debevec, Tina M. Iverson, Yangmei Li, Eun Jeong Cho, Nicole A. Perry, Chandra Bartholomeusz, and Andrea Piserchio
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,Tertiary amine ,Protein complex assembly ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,Humans ,Phosphorylation ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Protein Kinase Inhibitors ,Guanidine ,Mitogen-Activated Protein Kinase 1 ,Binding Sites ,Dose-Response Relationship, Drug ,biology ,010405 organic chemistry ,Chemistry ,Kinase ,Active site ,General Medicine ,0104 chemical sciences ,Cell biology ,Enzyme Activation ,030104 developmental biology ,Docking (molecular) ,biology.protein ,Molecular Medicine - Abstract
Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure–activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507–1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507–8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507–8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein–protein interactions.
- Published
- 2019
19. NMR solution structures of Runella slithyformis RNA 2'-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity
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Sébastien Alphonse, Stewart Shuman, Ankan Banerjee, Ranajeet Ghose, and Swathi Dantuluri
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Models, Molecular ,Stereochemistry ,Protein Conformation ,AcademicSubjects/SCI00010 ,Cytophagaceae ,NAR Breakthrough Article ,Biology ,010402 general chemistry ,Ligands ,01 natural sciences ,Phosphotransferase ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Bacterial Proteins ,Ribose ,Genetics ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Nicotinamide ,Adenosine diphosphate ribose ,Nucleotides ,Phosphotransferases ,RNA ,NAD ,0104 chemical sciences ,NAD binding ,chemistry ,Mutagenesis ,Nicotinamide riboside ,NAD+ kinase ,Protein Binding - Abstract
Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.
- Published
- 2021
20. Regulation of the catalytic activity of by-kinases
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Fatlum Hajredini and Ranajeet Ghose
- Subjects
Biophysics - Published
- 2022
21. The Cold-Unfolded State Is Expanded but Contains Long- and Medium-Range Contacts and Is Poorly Described by Homopolymer Models
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Natalie E. Stenzoski, Junjie Zou, Ranajeet Ghose, Alex S. Holehouse, Daniel P. Raleigh, and Andrea Piserchio
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Models, Molecular ,Ribosomal Proteins ,Quantitative Biology::Biomolecules ,0303 health sciences ,Protein Denaturation ,Protein Folding ,genetic structures ,Chemistry ,Quantitative Biology::Molecular Networks ,030302 biochemistry & molecular biology ,Thermodynamics ,State (functional analysis) ,Biochemistry ,Protein Structure, Tertiary ,Quantitative Biology::Subcellular Processes ,Cold Temperature ,03 medical and health sciences ,X-Ray Diffraction ,Medium range ,Scattering, Small Angle ,Point Mutation ,Hydrophobic and Hydrophilic Interactions ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Cold unfolding of proteins is predicted by the Gibbs-Helmholtz equation and is thought to be driven by a strongly temperature-dependent interaction of protein nonpolar groups with water. Studies of the cold-unfolded state provide insight into protein energetics, partially structured states, and folding cooperativity and are of practical interest in biotechnology. However, structural characterization of the cold-unfolded state is much less extensive than studies of thermally or chemically denatured unfolded states, in large part because the midpoint of the cold unfolding transition is usually below freezing. We exploit a rationally designed point mutation (I98A) in the hydrophobic core of the C-terminal domain of the ribosomal protein L9 that allows the cold denatured state ensemble to be observed above 0 °C at near neutral pH and ambient pressure in the absence of added denaturants. A combined approach consisting of paramagnetic relaxation enhancement measurements, analysis of small-angle X-ray scattering data, all-atom simulations, and polymer theory provides a detailed description of the cold-unfolded state. Despite a globally expanded ensemble, as determined by small-angle X-ray scattering, sequence-specific medium- and long-range interactions in the cold-unfolded state give rise to deviations from homopolymer-like behavior. Our results reveal that the cold-denatured state is heterogeneous with local and long-range intramolecular interactions that may prime the folded state and also demonstrate that significant long-range interactions are compatible with expanded unfolded ensembles. The work also highlights the limitations of homopolymer-based descriptions of unfolded states of proteins.
- Published
- 2020
22. Keep a lid on it: A troika in kinase allostery
- Author
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Ranajeet Ghose
- Subjects
0301 basic medicine ,Allosteric regulation ,Plasmodium falciparum ,Protozoan Proteins ,Molecular Dynamics Simulation ,Biochemistry ,03 medical and health sciences ,Allosteric Regulation ,Protein Domains ,Cyclic GMP-Dependent Protein Kinases ,Animals ,Editors' Picks ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Cyclic GMP ,Life Cycle Stages ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Kinase ,Cell Biology ,Surface Plasmon Resonance ,biology.organism_classification ,Recombinant Proteins ,Cell biology ,030104 developmental biology ,Editors' Picks Highlights ,Mutagenesis, Site-Directed ,cGMP-dependent protein kinase ,Function (biology) ,Protein Binding - Abstract
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum. Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog–bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive “mixed” intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.
- Published
- 2020
23. Long-range dynamic correlations regulate the catalytic activity of the bacterial tyrosine kinase Wzc
- Author
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Fatlum Hajredini, Ranajeet Ghose, and Andrea Piserchio
- Subjects
chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Futile cycle ,Escherichia coli Proteins ,Autophosphorylation ,Membrane Proteins ,Isothermal titration calorimetry ,Protein-Tyrosine Kinases ,010402 general chemistry ,01 natural sciences ,Oligomer ,0104 chemical sciences ,Adenosine Diphosphate ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,chemistry ,ATP hydrolysis ,Biophysics ,Escherichia coli ,Nucleotide ,Tyrosine ,Tyrosine kinase ,030304 developmental biology - Abstract
BY-kinases represent a highly conserved family of protein tyrosine kinases unique to bacteria without eukaryotic orthologs. BY-kinases are regulated by oligomerization-enabled transphosphorylation on a C-terminal tyrosine cluster through a process with sparse mechanistic detail. Using the catalytic domain (CD) of the archetypal BY-kinase, Escherichia coli Wzc, and enhanced-sampling molecular dynamics simulations, isothermal titration calorimetry and nuclear magnetic resonance measurements, we propose a mechanism for its activation and nucleotide exchange. We find that the monomeric Wzc CD preferentially populates states characterized by distortions at its oligomerization interfaces and by catalytic element conformations that allow high-affinity interactions with ADP but not with ATP·Mg2+ We propose that oligomer formation stabilizes the intermonomer interfaces and results in catalytic element conformations suitable for optimally engaging ATP·Mg2+, facilitating exchange with bound ADP. This sequence of events, oligomerization, i.e., substrate binding, before engaging ATP·Mg2+, facilitates optimal autophosphorylation by preventing a futile cycle of ATP hydrolysis.
- Published
- 2020
24. Methyl NMR spectroscopy: Measurement of dynamics in viral RNA-directed RNA polymerases
- Author
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Ranajeet Ghose and Sébastien Alphonse
- Subjects
0301 basic medicine ,Protein Conformation ,Kinetics ,Molecular Dynamics Simulation ,010402 general chemistry ,Methylation ,01 natural sciences ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,Turn (biochemistry) ,03 medical and health sciences ,Bacterial Proteins ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Polymerase ,Quantitative Biology::Biomolecules ,biology ,Chemistry ,Protein dynamics ,Relaxation (NMR) ,RNA ,Nuclear magnetic resonance spectroscopy ,RNA-Dependent RNA Polymerase ,Protein Structure, Tertiary ,0104 chemical sciences ,030104 developmental biology ,Chemical physics ,biology.protein ,RNA, Viral ,Function (biology) - Abstract
Measurement of nuclear spin relaxation provides a powerful approach to access information about biomolecular conformational dynamics over several orders of magnitude in timescale. In several cases this knowledge in combination with spatial information from three-dimensional structures yields unique insight into protein stability and the kinetics and thermodynamics of their interactions and function. However, due to intrinsic difficulties in studying large systems using solution state nuclear magnetic resonance (NMR) approaches, until recently these measurements were limited to small-to-medium-sized systems. However, the development of a wide range of novel strategies that allow the selective isotope labeling of methyl groups in proteins have allowed the exploitation of the unique relaxation properties of this spin-system. This has in turn enabled the extension of NMR approaches to high molecular weight proteins including a variety of enzymes and their complexes. Here, we recount our experiences in obtaining assignments of the methyl resonances for two representative members of a class of RNA-directed RNA polymerases (RdRps) encoded by bacteriophages of the Cystoviridae family. We demonstrate the utility of these methyl probes, limited in number for one case and more numerous for the other, to investigate the conformational dynamics of RdRps on the fast (ps-ns) and slow (μs-ms) timescales.
- Published
- 2018
25. Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2+-Calmodulin
- Author
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Rinat R. Abzalimov, Kevin N. Dalby, David H. Giles, Fatlum Hajredini, Kwangwoon Lee, Michael W. Clarkson, Nathan Will, and Ranajeet Ghose
- Subjects
Elongation Factor 2 Kinase ,0301 basic medicine ,Calmodulin ,Protein Conformation ,Ribosome ,Article ,03 medical and health sciences ,Structural Biology ,Humans ,Phosphorylation ,education ,Molecular Biology ,education.field_of_study ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Translation (biology) ,Elongation factor ,Kinetics ,030104 developmental biology ,Protein kinase domain ,biology.protein ,Biophysics ,Calcium ,Hydrogen–deuterium exchange ,Elongation Factor-2 Kinase - Abstract
Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM) activated α–kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca(2+) ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry (XLMS) and small angle X-ray scattering (SAXS) measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen deuterium exchange mass spectrometry (HXMS) and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.
- Published
- 2018
26. Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques
- Author
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Reza Khayat, Sébastien Alphonse, Boris Itin, and Ranajeet Ghose
- Subjects
Polyproteins ,biology ,Chemistry ,Virus Assembly ,viruses ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Biochemistry ,In vitro ,Protein expression ,Bacteriophage phi 6 ,Bacteriophage ,Viral Proteins ,Capsid ,Microscopy, Electron, Transmission ,Solid-state nuclear magnetic resonance ,Biophysics ,Nuclear Magnetic Resonance, Biomolecular - Abstract
While solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for studying viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multiprotein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses. In vitro capsid assembly by combining individually purified, labeled, and unlabeled components in NMR quantities is not a viable option for most viruses. To overcome this barrier, we present a method that utilizes sequential protein expression and in cell assembly of component-specifically labeled viral capsids in amounts suitable for NMR studies. We apply this approach to purify capsids of bacteriophage ϕ6 isotopically labeled on only one of its four constituent protein components, the NTPase P4. Using P4-labeled ϕ6 capsids and the sensitivity enhancement provided by dynamic nuclear polarization, we illustrate the utility of this method to enable ssNMR studies of complex viruses.
- Published
- 2018
27. Characterization of DNA Binding by the Isolated N-Terminal Domain of Vaccinia Virus DNA Topoisomerase IB
- Author
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Benjamin Reed, Lyudmila Yakovleva, Stewart Shuman, and Ranajeet Ghose
- Subjects
Models, Molecular ,0301 basic medicine ,HMG-box ,Stereochemistry ,Recombinant Fusion Proteins ,Vaccinia virus ,Calorimetry ,Biochemistry ,Article ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Interaction Domains and Motifs ,Nucleotide Motifs ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,DNA ligase ,030102 biochemistry & molecular biology ,biology ,Topoisomerase ,Osmolar Concentration ,Titrimetry ,Isothermal titration calorimetry ,DNA ,DNA-binding domain ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Isoenzymes ,Kinetics ,030104 developmental biology ,Amino Acid Substitution ,DNA Topoisomerases, Type I ,chemistry ,Mutation ,Phosphodiester bond ,Hydrodynamics ,biology.protein ,DNA supercoil - Abstract
Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5’-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3’-phosphotyrosyl(274))-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with lower cleavage site affinity compared to the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ~2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution NMR methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB are underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.
- Published
- 2017
28. Signal Integration at Elongation Factor 2 Kinase
- Author
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Scarlett B. Ferguson, David H. Giles, Rebecca M. Wellmann, Kevin N. Dalby, Tamer S. Kaoud, Gabriel Stancu, Ranajeet Ghose, Catrina A. Chitjian, and Clint D.J. Tavares
- Subjects
0301 basic medicine ,education.field_of_study ,Calmodulin ,Autophosphorylation ,Allosteric regulation ,Regulatory site ,Cell Biology ,Biology ,Biochemistry ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Protein kinase domain ,Ca2+/calmodulin-dependent protein kinase ,biology.protein ,Phosphorylation ,Elongation Factor-2 Kinase ,education ,Molecular Biology - Abstract
Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.
- Published
- 2017
29. Protein Structure and Interactions from Nuclear Magnetic Resonance Spectroscopy
- Author
-
Ranajeet Ghose
- Subjects
Nuclear magnetic resonance ,Protein structure ,Materials science ,Biomolecular complex ,Triple-resonance nuclear magnetic resonance spectroscopy ,Nuclear magnetic resonance spectroscopy of nucleic acids ,Nuclear magnetic resonance spectroscopy ,Fluorine-19 NMR - Published
- 2017
30. Cover Image, Volume 58, Issue 9
- Author
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Rachel M. Sammons, Ranajeet Ghose, Kenneth Y. Tsai, and Kevin N. Dalby
- Subjects
Cancer Research ,Molecular Biology - Published
- 2019
31. Allosteric Regulation of the Activity of BY-Kinases, a Unique Family of Bacterial Protein Tyrosine Kinases
- Author
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Rinat R. Abzalimov, Andrea Piserchio, Ranajeet Ghose, and Fatlum Hajredini
- Subjects
Bacterial protein ,Biochemistry ,Chemistry ,Kinase ,Allosteric regulation ,Biophysics ,Tyrosine kinase - Published
- 2021
32. Regulation of the Activity of Bacterial Tyrosine Kinases
- Author
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Ranajeet Ghose, Fatlum Hajredini, Andrea Piserchio, and Rinat R. Abzalimov
- Subjects
Biochemistry ,Chemistry ,Biophysics ,Tyrosine kinase - Published
- 2020
33. Nature of the Pre-Chemistry Ensemble in Mitogen-Activated Protein Kinases
- Author
-
Ranajeet Ghose
- Subjects
MAPK/ERK pathway ,0303 health sciences ,Kinase ,Chemistry ,MAP Kinase Signaling System ,Protein Conformation ,Protein dynamics ,Mitogen-activated protein ,03 medical and health sciences ,0302 clinical medicine ,Adenosine Triphosphate ,Structural Biology ,Docking (molecular) ,Biophysics ,Phosphorylation ,Humans ,Mitogen-Activated Protein Kinases ,Molecular Biology ,Ternary complex ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
In spite of the availability of a significant amount of structural detail on docking interactions involving mitogen-activated protein kinases (MAPKs) and their substrates, the mechanism by which the disordered phospho-acceptor on the substrate transiently interacts with the kinase catalytic elements and is phosphorylated, often with high efficiency, remains poorly understood. Here, this dynamic interaction is analyzed in the context of available biophysical and biochemical data for ERK2, an archetypal MAPK. A hypothesis about the nature of the ternary complex involving a MAPK, its substrate, and ATP immediately prior to the chemical step (the pre-chemistry complex) is proposed. It is postulated that the solution ensemble (the pre-chemistry ensemble) representing the pre-chemistry complex comprises several conformations that are linked by dynamics on multiple timescales. These individual conformations possess different intrinsic abilities to proceed through the chemical step. The overall rate of chemistry is therefore related to the microscopic nature of the pre-chemistry ensemble, its constituent conformational microstates, and their intrinsic abilities to yield a phosphorylated product. While characterizing these microstates within the pre-chemistry ensemble in atomic or near-atomic detail is an extremely challenging proposition, recent developments in hybrid methodologies that employ computational approaches driven by experimental data appear to provide the most promising path forward toward achieving this goal.
- Published
- 2018
34. Protein NMR
- Author
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Ranajeet Ghose
- Published
- 2018
- Full Text
- View/download PDF
35. Protein Unfolded States are Characterized by the Duality of Sequence-Specific Conformational Preferences and Ensemble-Averaged Features of Canonical Random Coils
- Author
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Ivan Peran, Natalie E. Stenzoski, Daniel P. Raleigh, Junjie Zou, Isaac S. Carrico, Ranajeet Ghose, Alex S. Holehouse, Andrea Piserchio, Rohit V. Pappu, and Osman Bilsel
- Subjects
Pure mathematics ,Biophysics ,Duality (optimization) ,Sequence (medicine) ,Mathematics - Published
- 2019
36. Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2
- Author
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Tamer S. Kaoud, Kevin N. Dalby, Kari Callaway, Andrea Piserchio, Mangalika Warthaka, and Ranajeet Ghose
- Subjects
0301 basic medicine ,MAP Kinase Signaling System ,Protein Conformation ,Plasma protein binding ,Catalysis ,Proto-Oncogene Protein c-ets-1 ,03 medical and health sciences ,Protein structure ,Humans ,Binding site ,Phosphorylation ,Transcription factor ,Nuclear Magnetic Resonance, Biomolecular ,Mitogen-Activated Protein Kinase 1 ,Multidisciplinary ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Kinase ,Phosphoproteins ,030104 developmental biology ,Biochemistry ,PNAS Plus ,Docking (molecular) ,Mitogen-activated protein kinase ,Biophysics ,biology.protein ,Apoptosis Regulatory Proteins ,Protein Binding - Abstract
Mitogen-activated protein (MAP) kinase substrates are believed to require consensus docking motifs (D-site, F-site) to engage and facilitate efficient site-specific phosphorylation at specific serine/threonine-proline sequences by their cognate kinases. In contrast to other MAP kinase substrates, the transcription factor Ets-1 has no canonical docking motifs, yet it is efficiently phosphorylated by the MAP kinase ERK2 at a consensus threonine site (T38). Using NMR methodology, we demonstrate that this phosphorylation is enabled by a unique bipartite mode of ERK2 engagement by Ets-1 and involves two suboptimal noncanonical docking interactions instead of a single canonical docking motif. The N terminus of Ets-1 interacts with a part of the ERK2 D-recruitment site that normally accommodates the hydrophobic sidechains of a canonical D-site, retaining a significant degree of disorder in its ERK2-bound state. In contrast, the C-terminal region of Ets-1, including its Pointed (PNT) domain, engages in a largely rigid body interaction with a section of the ERK2 F-recruitment site through a binding mode that deviates significantly from that of a canonical F-site. This latter interaction is notable for the destabilization of a flexible helix that bridges the phospho-acceptor site to the rigid PNT domain. These two spatially distinct, individually weak docking interactions facilitate the highly specific recognition of ERK2 by Ets-1, and enable the optimal localization of its dynamic phospho-acceptor T38 at the kinase active site to enable efficient phosphorylation.
- Published
- 2017
37. Cystoviral RNA-directed RNA Polymerases: Regulation of RNA Synthesis on Multiple Time and Length Scales
- Author
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Ranajeet Ghose and Sébastien Alphonse
- Subjects
0301 basic medicine ,Cystoviridae ,Gene Expression Regulation, Viral ,Models, Molecular ,Cancer Research ,Time Factors ,Transcription, Genetic ,Protein Conformation ,Computational biology ,Virus Replication ,Cystovirus ,Article ,Bacteriophage ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Virology ,RNA polymerase ,Polymerase ,biology ,RNA ,DNA-Directed RNA Polymerases ,biology.organism_classification ,Molecular biology ,030104 developmental biology ,Infectious Diseases ,chemistry ,Polynucleotide ,RNA editing ,biology.protein - Abstract
P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family. These range from atomic resolution snapshots of P2 trapped in functionally significant states, in complex with catalytic/structural metal ions, polynucleotide templates and substrate nucleoside triphosphates, to P2 in the context of viral capsids providing structural insight into the assembly of supramolecular complexes and regulatory interactions therein. They include in vitro biochemical studies using P2 purified to homogeneity and in vivo studies utilizing infectious core particles. Recent advances in experimental techniques have also allowed access to the temporal dimension and enabled the characterization of dynamics of P2 on the sub-nanosecond to millisecond timescale through measurements of nuclear spin relaxation in solution and single molecule studies of transcription from seconds to minutes. Below we summarize the most significant results that provide critical insight into the role of P2 in regulating RNA synthesis in cystoviruses.
- Published
- 2017
38. Abstract 3872: Targeting multi-functional ERK-protein complexes in vivo
- Author
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Andrea Piserchio, Jacey R. Pridgen, Micael Cano, Nancy D. Ebelt, Mangalika Warthaka, Eric V. Anslyn, William H. Johnson, Diana Zamora-Olivares, Tamer S. Kaoud, Kenneth Y. Tsai, Kevin N. Dalby, Sabrina X. Van Ravenstein, Rachel M. Sammons, Ramakrishna Edupuganti, Ranajeet Ghose, and Pengyu Ren
- Subjects
MAPK/ERK pathway ,Cancer Research ,Melanoma ,HEK 293 cells ,Mutant ,Tumor initiation ,Biology ,medicine.disease ,chemistry.chemical_compound ,Oncology ,chemistry ,In vivo ,Apoptosis ,Cancer research ,medicine ,Growth inhibition - Abstract
Mutations in pathways that enhance the activity of ERK1 and ERK2 are frequently present in human cancers, reflecting their important roles in tumor initiation, and progression. This notion is reinforced by observations in BRAF V600E mutant melanoma where the majority of the mechanisms of resistance to FDA-approved combination therapies targeting BRAF and MEK involve reactivation of ERK1 and ERK2. Recently, the direct targeting of the ERK enzymes using ATP-competitive inhibitors has emerged as an attractive strategy to overcome acquired resistance to current combination therapies. The ERK enzymes employ unique mechanisms of molecular recognition to engage protein components of the MAPK pathway. Here we report the potent targeting of an ERK-protein docking interaction by a small molecule thiotriazole, which abrogates ERK signaling in vivo. The thiotriazole binds covalently to a highly conserved cysteine residue within the D-recruitment site of ERK1/2 with more than a 100-fold discrimination over other MAPKs (e.g. JNK1/2, p38MAPKs and ERK5). Treatment of various BRAF-inhibitor naive or inhibitor-resistant melanoma cell lines expressing BRAF V600E with the thiotriazole for 2 hours induces dose-dependent inhibition of ERK activation and downstream signaling. Inhibition is maintained for up to 5 hours following thiotriazole washout and induces apoptosis and growth inhibition. Treatment of mice carrying a BRAFV600E A375 melanoma xenograft with the thiotriazole blocked tumor growth. Transient expression of a mutant form of ERK2, which is resistant to the thiotriazole, promotes survival of A375 and HEK 293 cells treated with thiotriazole. This study lays the foundation for developing a new modality for the treatment of solid tumors driven by excessive ERK signaling. Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Diana Zamora-Olivares, Sabrina V. Ravenstein, Jacey R. Pridgen, Ramakrishna Edupuganti, Rachel Sammons, Micael Cano, Mangalika Warthaka, Pengyu Ren, Eric V. Anslyn, Kenneth Y. Tsai, Ranajeet Ghose, Kevin N. Dalby. Targeting multi-functional ERK-protein complexes in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3872.
- Published
- 2019
39. Structure of the RNA-directed RNA Polymerase from the cystovirus ϕ12
- Author
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Ranajeet Ghose, Zhen Ren, and Matthew C. Franklin
- Subjects
biology ,Stereochemistry ,RNA-dependent RNA polymerase ,Active site ,biology.organism_classification ,Biochemistry ,Cystovirus ,Bacteriophage ,A-site ,Crystallography ,RNA-Directed RNA Polymerase ,Structural Biology ,Transcription (biology) ,biology.protein ,Transferase ,Molecular Biology - Abstract
We have determined the structure of P2, the self-priming RdRp from cystovirus φ12 in two crystal forms (A, B) at resolutions of 1.7 A and 2.1 A. Form A contains Mg(2+) bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts-mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo.
- Published
- 2013
40. A Measured Approach: Determining the PLCγ1 Docking Site on Itk Using a Biochemical Ruler
- Author
-
Ranajeet Ghose
- Subjects
chemistry.chemical_classification ,Kinase ,Biology ,Phospholipase C gamma ,SH2 domain ,Amino acid ,Biochemistry ,chemistry ,Structural Biology ,Docking (molecular) ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Tyrosine kinase - Abstract
In this issue of the Journal of Molecular Biology Xie et. al. describe the interaction between IL-2 inducible T-cell kinase (Itk), a non-receptor tyrosine kinase, and its substrate PLCγ1. The authors demonstrate that a previously determined set of basic amino acid residues1 on the C-terminal SH2 domain of PLCγ1, interacts with an acidic patch on the G helix (αG) of the catalytic domain of Itk. The results described here reinforce the importance of αG as a mediator of intermolecular interactions in protein kinases.
- Published
- 2013
41. Sequence-specific backbone 1H, 13C and 15N assignments of the catalytic domain of the Escherichia coli protein tyrosine kinase, Wzc
- Author
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Kaushik Dutta, Ranajeet Ghose, and Deniz B. Temel
- Subjects
Protein tyrosine phosphatase ,Biology ,medicine.disease_cause ,SH2 domain ,Biochemistry ,Receptor tyrosine kinase ,SH3 domain ,Structural Biology ,medicine ,biology.protein ,Phosphorylation ,Tyrosine kinase ,Escherichia coli ,Peptide sequence - Abstract
Protein tyrosine kinases in bacteria are structurally and functionally distinct from their eukaryotic counterparts. The largest family of bacterial tyrosine kinases, the BY-kinase family, is highly conserved in Gram-negative and Gram-positive species, and plays a central role in biofilm and capsule formation. In Escherichia coli the BY-kinase, Wzc, is a critical component of the machinery responsible for the synthesis and export of the exo-polysaccharide colanic acid, a key constituent of biofilms. Here we present the main-chain (1)H(N), (15)N, (13)C' and (13)Cα, side-chain (13)Cβ resonance assignments for a construct that encodes the entire 274-residue cytosolic catalytic domain of Wzc.
- Published
- 2012
42. Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase
- Author
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David H. Giles, Ranajeet Ghose, Scarlet B. Ferguson, Andrea Piserchio, Isaac Snyder, Nathan Will, and Kevin N. Dalby
- Subjects
0301 basic medicine ,Elongation Factor 2 Kinase ,education.field_of_study ,Protein Conformation ,Substrate (chemistry) ,Biology ,Biochemistry ,Ribosome ,Article ,Elongation factor ,03 medical and health sciences ,Tetratricopeptide ,Crystallography ,030104 developmental biology ,Protein structure ,Biophysics ,Phosphorylation ,Transferase ,Animals ,Elongation Factor-2 Kinase ,education ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, which is predicted to contain several SEL-1-like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution nuclear magnetic resonance methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (αI) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (αII-αIV). The helical core is structurally similar to members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, αII and αIII, comprise the TPR, and the last helix, αIV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing αIV and, therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding nor isolated eEF-2K627-725 inhibits eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.
- Published
- 2016
43. Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION
- Author
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Clint D J, Tavares, David H, Giles, Gabriel, Stancu, Catrina A, Chitjian, Scarlett B, Ferguson, Rebecca M, Wellmann, Tamer S, Kaoud, Ranajeet, Ghose, and Kevin N, Dalby
- Subjects
Elongation Factor 2 Kinase ,Amino Acid Substitution ,Calmodulin ,Cell Line, Tumor ,Mutation, Missense ,Serine ,Enzymology ,Humans ,Calcium ,Phosphorylation - Abstract
Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.
- Published
- 2016
44. Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin
- Author
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Sébastien Alphonse, Ranajeet Ghose, David H. Giles, Kwangwoon Lee, Clint D.J. Tavares, Rebecca M. Wellmann, Andrea Piserchio, and Kevin N. Dalby
- Subjects
0301 basic medicine ,Elongation Factor 2 Kinase ,Models, Molecular ,Protein Conformation, alpha-Helical ,Calmodulin ,Static Electricity ,Gene Expression ,Plasma protein binding ,Crystallography, X-Ray ,Substrate Specificity ,03 medical and health sciences ,Protein structure ,Structural Biology ,Cell Line, Tumor ,Protein biosynthesis ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Binding site ,Phosphorylation ,education ,Molecular Biology ,Peptide sequence ,education.field_of_study ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Epithelial Cells ,Peptide Elongation Factors ,Recombinant Proteins ,030104 developmental biology ,Biochemistry ,biology.protein ,Biophysics ,Thermodynamics ,Calcium ,Elongation Factor-2 Kinase ,Peptides ,Hydrophobic and Hydrophilic Interactions ,Protein Binding - Abstract
Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells.
- Published
- 2016
45. Sequence-specific 1H, 13C and 15N assignments of the phosphoesterase (PE) domain of Pseudomonas aeruginosa DNA ligase D (LigD)
- Author
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Aswin Natarajan, Ranajeet Ghose, Kaushik Dutta, Pravin A. Nair, and Stewart Shuman
- Subjects
DNA Ligases ,DNA Repair ,DNA repair ,medicine.disease_cause ,Biochemistry ,Article ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,medicine ,Nuclear Magnetic Resonance, Biomolecular ,Polymerase ,chemistry.chemical_classification ,Carbon Isotopes ,DNA ligase ,Nitrogen Isotopes ,biology ,Pseudomonas aeruginosa ,Phosphoric Monoester Hydrolases ,Protein Structure, Tertiary ,Non-homologous end joining ,Enzyme ,chemistry ,biology.protein ,DNA - Abstract
DNA ligase D (LigD), consisting of polymerase, ligase and phosphoesterase domains, is the essential catalyst of the bacterial non-homologous end-joining pathway of DNA double-strand break repair. The phosphoesterase (PE) module performs manganese-dependent 3'-phosphomonoesterase and 3'-ribonucleoside resection reactions that heal broken ends in preparation for sealing. LigD PE exemplifies a structurally and mechanistically unique class of DNA end-processing enzymes. Here, we present the resonance assignments of the PE domain of Pseudomonas aeruginosa LigD comprising the N-terminal 177 residues.
- Published
- 2011
46. On the measurement of 15N–{1H} nuclear Overhauser effects. 2. Effects of the saturation scheme and water signal suppression
- Author
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Shibani Battacharya, Ranajeet Ghose, Amy Reichel, Fabien Ferrage, and David Cowburn
- Subjects
0303 health sciences ,Nuclear and High Energy Physics ,Chemistry ,Protein dynamics ,Biophysics ,Nuclear magnetic resonance spectroscopy ,Nuclear Overhauser effect ,Radiation ,010402 general chemistry ,Condensed Matter Physics ,Residual ,01 natural sciences ,Biochemistry ,Signal ,0104 chemical sciences ,03 medical and health sciences ,Nuclear magnetic resonance ,Radiation damping ,Atomic physics ,Saturation (chemistry) ,030304 developmental biology - Abstract
Measurement of steady-state (15)N-{(1)H} nuclear Overhauser effects forms a cornerstone of most methods to determine protein backbone dynamics from spin-relaxation data, since it is the most reliable probe of very fast motions on the ps-ns timescale. We have, in two previous publications (J. Magn. Reson. 192 (2008) 302-313; J. Am. Chem. Soc. 131 (2009) 6048-6049) reevaluated spin-dynamics during steady-state (or "saturated") and reference experiments, both of which are required to determine the NOE ratio. Here we assess the performance of several windowed and windowless sequences to achieve effective saturation of protons in steady-state experiments. We also evaluate the influence of the residual water signal due to radiation damping on the NOE ratio. We suggest a recipe that allows one to determine steady-state (15)N-{(1)H} NOE's without artifacts and with the highest possible accuracy.
- Published
- 2010
47. Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies
- Author
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Ranajeet Ghose, David Cowburn, and Andrea Piserchio
- Subjects
Chemistry ,Proto-Oncogene Proteins pp60(c-src) ,Phosphatase ,medicine.disease_cause ,Biochemistry ,Article ,Recombinant Proteins ,Catalysis ,Domain (software engineering) ,law.invention ,src Homology Domains ,law ,Escherichia coli ,medicine ,Recombinant DNA ,Animals ,Phosphorylation ,Chickens ,Nuclear Magnetic Resonance, Biomolecular ,Tyrosine kinase ,Spectroscopy ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs), making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain, a complete understanding of its catalytic regulation is unavailable. Also unavailable are atomic or near-atomic resolution information about their interactions, often weak or transient, with regulating phosphatases and downstream targets. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. Through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, we have achieved the ability to bacterially produce large quantities of the isotopically-labeled catalytic domain of c-Src, the prototypical SFK, and of its activating Tyr-phosphorylated form. All constructs produce excellent spectra allowing solution NMR studies of this family in an efficient manner.
- Published
- 2009
48. Sequence-specific 1HN, 13C, and 15N backbone resonance assignments of the 34 kDa Paramecium bursaria Chlorella virus 1 (PBCV1) DNA ligase
- Author
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Ranajeet Ghose, Andrea Piserchio, Stewart Shuman, and Pravin A. Nair
- Subjects
Magnetic Resonance Spectroscopy ,Paramecium ,DNA Ligases ,Molecular Sequence Data ,Biochemistry ,Article ,Viral Proteins ,chemistry.chemical_compound ,Structural Biology ,Animals ,A-DNA ,Amino Acid Sequence ,Peptide sequence ,chemistry.chemical_classification ,Carbon Isotopes ,DNA ligase ,Nitrogen Isotopes ,biology ,Nuclear magnetic resonance spectroscopy ,Nucleotidyltransferase ,biology.organism_classification ,Molecular Weight ,chemistry ,Protons ,DNA - Abstract
Chlorella virus DNA ligase (ChVLig) is a minimal (298-amino acid) pluripotent ATP-dependent ligase composed of three structural modules--a nucleotidyltransferase domain, an OB domain, and a beta-hairpin latch--that forms a circumferential clamp around nicked DNA. ChVLig provides an instructive model to understand the chemical and conformational steps of nick repair. Here we report the assignment of backbone (13)C, (15)N, (1)H(N) resonances of this 34.2 kDa protein, the first for a DNA ligase in full-length form.
- Published
- 2009
49. Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-directed RNA Polymerase
- Author
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Sébastien Alphonse, Shibani Bhattacharya, Ranajeet Ghose, and Haiyan Wang
- Subjects
Cystoviridae ,Protein Conformation ,In silico ,Molecular Sequence Data ,RNA-dependent RNA polymerase ,Molecular Dynamics Simulation ,Biochemistry ,Methylation ,Cystovirus ,Article ,chemistry.chemical_compound ,Viral Proteins ,Protein structure ,RNA polymerase ,Point Mutation ,Amino Acid Sequence ,Polymerase ,biology ,Point mutation ,RNA ,biology.organism_classification ,RNA-Dependent RNA Polymerase ,Molecular biology ,chemistry ,biology.protein ,Biophysics ,Guanosine Triphosphate - Abstract
Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.
- Published
- 2015
50. Erratum: Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2
- Author
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Andrea Piserchio, Venkatesh Ramakrishan, Hsin Wang, Tamer S. Kaoud, Boris Arshava, Kaushik Dutta, Kevin N. Dalby, and Ranajeet Ghose
- Subjects
Mitogen-Activated Protein Kinase 1 ,Models, Molecular ,Binding Sites ,Multidisciplinary ,Protein Conformation ,Catalytic Domain ,Humans ,DNA ,Erratum ,Phosphorylation ,ets-Domain Protein Elk-1 - Abstract
The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS.
- Published
- 2015
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