Search

The intrinsically disordered protein α-synuclein (aSN) is, in its fibrillated state, the main component of Lewy bodies-hallmarks of Parkinson's disease. Additional Lewy body components include glycosaminoglycans, including heparan sulfate proteoglycans. In humans, heparan sulfate has, in an age-dependent manner, shown increased levels of sulfation. Heparin, a highly sulfated glycosaminoglycan, is a relevant mimic for mature heparan sulfate and has been shown to influence aSN fibrillation. Here, we decompose the underlying properties of the interaction between heparin and aSN and the effect of heparin on fibrillation. Via the isolation of the first 61 residues of aSN, which lacked intrinsic fibrillation propensity, fibrillation could be induced by heparin, and access to the initial steps in fibrillation was possible. Here, structural changes with shifts from disorder via type I β-turns to β-sheets were revealed, correlating with an increase in the aSN 1-61 /heparin molar ratio. Fluorescence microscopy revealed that heparin and aSN 1-61 co-exist in the final fibrils. We conclude that heparin can induce the fibrillation of aSN 1-61 , through binding to the N-terminal with an affinity that is higher in the truncated form of aSN. It does so by specifically modulating the structure of aSN via the formation of type I β-turn structures likely critical for triggering aSN fibrillation.

Living organisms depend on timely and organized interactions between proteins linked in interactomes of high complexity. The recent increased precision by which protein interactions can be studied, and the enclosure of intrinsic structural disorder, suggest that it is time to zoom out and embrace protein interactions beyond the most central points of physical encounter. The present paper discusses protein-protein interactions in the view of structural disorder with an emphasis on flanking regions and contexts of disorder-based interactions. Context constitutes an overarching concept being of physicochemical, biomolecular, and physiological nature, but it also includes the immediate molecular context of the interaction. For intrinsically disordered proteins, which often function by exploiting short linear motifs, context contributes in highly regulatory and decisive manners and constitute a yet largely unrecognized source of interaction potential in a multitude of biological processes. Through selected examples, this review emphasizes how multivalency, charges and charge clusters, hydrophobic patches, dynamics, energetic frustration, and ensemble redistribution of flanking regions or disordered contexts are emerging as important contributors to allosteric regulation, positive and negative cooperativity, feedback regulation and negative selection in binding. The review emphasizes that understanding context, and in particular the role the molecular disordered context and flanking regions take on in protein interactions, constitute an untapped well of energetic modulation potential, also of relevance to drug discovery and development., (Copyright © 2020 Bugge, Brakti, Fernandes, Dreier, Lundsgaard, Olsen, Skriver and Kragelund.)

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Structural data on membrane proteins in a lipid membrane environment is challenging to obtain but needed to provide information on the, often essential, protein-lipid interplay. A common experimental bottleneck in obtaining such data is providing samples in sufficient amounts and quality required for structural studies. We developed a new production protocol for the single-pass transmembrane protein (SPTMP) tissue factor (TF), exploiting the high expression level in E. coli inclusion bodies and subsequent refolding. This provided more than 5 mg of functional TF per liter bacterial culture. This is substantially more than what was obtained by the classical approaches for expressing TF in the membrane-anchored configuration. We optimized reconstitution into circularized nanodiscs enabling the formation of stable, TF loaded nanodiscs with different lipid compositions and with a limited material waste. The blood coagulation cascade is initiated by the complex formation between TF and Factor VIIa (FVIIa), and we probed this interaction by a functional assay and SPR measurements, which revealed similar activity and binding kinetics as TF produced by other protocols, demonstrating that high-yield production does not compromise TF function. Furthermore, the amounts of sample produced permitted initial small angle X-ray scattering studies providing the first structural information about TF and its binding to FVIIa in a lipid environment. This strategy possibly allows for probing the multicomponent complex TF:FVIIa together with its substrate Factor X on a lipid bilayer, but may also be relevant as a production strategy for other SPTMP for which structural information, in general, is limited., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

In the brain, α-synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co-structures, and aSN in mammalian cells. Enabled by the isolation of the membrane-bound state, we show that within the previously described N-terminal membrane anchor, membrane interaction relies both on an N-terminal tail (NTT) head group layer insertion of 14 residues and a folded-upon-binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co-structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N-terminal anchor couples N-terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Life is completely dependent on water. To analyze the role of water as a solvent in biology, we replaced water with heavy water (D 2 O) and investigated the biological effects by a wide range of techniques, using Schizosaccharomyces pombe as model organism. We show that high concentrations of D 2 O lead to altered glucose metabolism and growth retardation. After prolonged incubation in D 2 O, cells displayed gross morphological changes, thickened cell walls, and aberrant cytoskeletal organization. By transcriptomics and genetic screens, we show that the solvent replacement activates two signaling pathways: (1) the heat-shock response pathway and (2) the cell integrity pathway . Although the heat-shock response system upregulates various chaperones and other stress-relieving enzymes, we find that the activation of this pathway does not offer any fitness advantage to the cells under the solvent-replaced conditions. However, limiting the D 2 O-triggered activation of the cell integrity pathway allows cell growth when H 2 O is completely replaced with D 2 O. The isolated D 2 O-tolerant strains may aid biological production of deuterated biomolecules.

Protein domains constitute regions of distinct structural properties and molecular functions that are retained when removed from the rest of the protein. However, due to the lack of tertiary structure, the identification of domains has been largely neglected for long (>50 residues) intrinsically disordered regions. Here we present a sequence-based approach to assess and visualize domain organization in long intrinsically disordered regions based on compositional sequence biases. An online tool to find putative intrinsically disordered domains (IDDomainSpotter) in any protein sequence or sequence alignment using any particular sequence trait is available at http://www.bio.ku.dk/sbinlab/IDDomainSpotter. Using this tool, we have identified a putative domain enriched in hydrophilic and disorder-promoting residues (Pro, Ser, and Thr) and depleted in positive charges (Arg and Lys) bordering the folded DNA-binding domains of several transcription factors (p53, GCR, NAC46, MYB28, and MYB29). This domain, from two different MYB transcription factors, was characterized biophysically to determine its properties. Our analyses show the domain to be extended, dynamic and highly disordered. It connects the DNA-binding domain to other disordered domains and is present and conserved in several transcription factors from different families and domains of life. This example illustrates the potential of IDDomainSpotter to predict, from sequence alone, putative domains of functional interest in otherwise uncharacterized disordered proteins., (© 2019 The Protein Society.)

A hallmark of Parkinson's disease is the presence of Lewy bodies consisting of lipids and proteins, mainly fibrillated α-synuclein (aSN). aSN is an intrinsically disordered protein exerting its physiological role in an ensemble of states, one of which coexists in large assemblies with lipids, recently termed co-structures. Here, we decipher the kinetics of aSN:lipid co-structure formation to decode its mechanism of formation, and we show that the co-structures form with a distinct stoichiometry. Through seeded fibrillation assays, we demonstrate that aSN:lipid co-structures accelerate aSN fibril nucleation compared to lipid vesicles alone. A small-angle X-ray scattering-based model is proposed in which aSN decorates the lipid vesicle surface, yielding properties similar to those of the fibril surface, enhancing fibril nucleation. The delicate balance of aSN structural states close to and on the membrane may under given conditions, e.g., increased local concentrations, be a crucial switching factor between functional and pathological behavior.

Radical-Induced Cell Death1 (RCD1) functions as a cellular hub interacting with intrinsically disordered transcription factor regions, which lack a well-defined three-dimensional structure, to regulate plant stress. Here, we address the molecular evolution of the RCD1-interactome. Using bioinformatics, its history was traced back more than 480 million years to the emergence of land plants with the RCD1-binding short linear motif (SLiM) identified from mosses to flowering plants. SLiM variants were biophysically verified to be functional and to depend on the same RCD1 residues as the DREB2A transcription factor. Based on this, numerous additional members may be assigned to the RCD1-interactome. Conservation was further strengthened by similar intrinsic disorder profiles of the transcription factor homologs. The unique structural plasticity of the RCD1-interactome, with RCD1-binding induced α-helix formation in DREB2A, but not detectable in ANAC046 or ANAC013, is apparently conserved. Thermodynamic analysis also indicated conservation with interchangeability between Arabidopsis and soybean RCD1 and DREB2A, although with fine-tuned co-evolved binding interfaces. Interruption of conservation was observed, as moss DREB2 lacked the SLiM, likely reflecting differences in plant stress responses. This whole-interactome study uncovers principles of the evolution of SLiM:hub-interactions, such as conservation of α-helix propensities, which may be paradigmatic for disorder-based interactomes in eukaryotes.

Phosphorylation is one of the main regulators of cellular signaling typically occurring in flexible parts of folded proteins and in intrinsically disordered regions. It can have distinct effects on the chemical environment as well as on the structural properties near the modification site. Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins (IDPs) and the reliability of the analysis depends on an appropriate choice of random coil model. Random coil chemical shifts and sequence correction factors were previously determined for an Ac-QQXQQ-NH 2 -peptide series with X being any of the 20 common amino acids. However, a matching dataset on the phosphorylated states has so far only been incompletely determined or determined only at a single pH value. Here we extend the database by the addition of the random coil chemical shifts of the phosphorylated states of serine, threonine and tyrosine measured over a range of pH values covering the pKas of the phosphates and at several temperatures (www.bio.ku.dk/sbinlab/randomcoil). The combined results allow for accurate random coil chemical shift determination of phosphorylated regions at any pH and temperature, minimizing systematic biases of the secondary chemical shifts. Comparison of chemical shifts using random coil sets with and without inclusion of the phosphoryl group, revealed under/over estimations of helicity of up to 33%. The expanded set of random coil values will improve the reliability in detection and quantification of transient secondary structure in phosphorylation-modified IDPs.

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.

Physical interactions between members of the MYB and bHLH transcription factor (TF) families regulate many important biological processes in plants. Not all reported MYB-bHLH interactions can be explained by the known binding sites in the R3 repeat of the MYB DNA-binding domain. Noteworthy, most of the sequence diversity of MYB TFs lies in their non-MYB regions, which contain orphan small subgroup-defining motifs not yet linked to molecular functions. Here, we identified the motif mediating interaction between MYB TFs from subgroup 12 and their bHLH partners. Unlike other known MYB-bHLH interactions, the motif locates to the centre of the predicted disordered non-MYB region. We characterised the core motif, which enabled accurate prediction of previously unknown bHLH-interacting MYB TFs in Arabidopsis thaliana, and we confirmed its functional importance in planta. Our results indicate a correlation between the MYB-bHLH interaction affinity and the phenotypic output controlled by the TF complex. The identification of an interaction motif outside R3 indicates that MYB-bHLH interactions must have arisen multiple times, independently and suggests many more motifs of functional relevance to be harvested from subgroup-specific studies., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Several transcription factor (TF) families, including the MYB family, regulate a wide array of biological processes. TFs contain DNA-binding domains (DBDs) and regulatory regions; although information on protein structure is scarce for plant MYB TFs, various in silico methods suggest that the non-MYB regions contain extensive intrinsically disordered regions (IDRs). Although IDRs do not fold into stable globular structures, they comprise functional regions including interaction motifs, and recent research has shown that IDRs perform crucial biological roles. We map here domain organization, disorder predictions, and functional regions across the entire Arabidopsis thaliana R2R3 MYB TF family, and highlight where an increased research focus will be necessary to shape a new understanding of structure-function relationships in plant TFs., (Copyright © 2019. Published by Elsevier Ltd.)

Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na + /H + -exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer pThr over pSer, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of pT779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.

The erythropoietin receptor (EPOR) plays an essential role in erythropoiesis and other cellular processes by forming distinct signaling complexes composed of EPOR homodimers or hetero-oligomers between the EPOR and another receptor, but the mechanism of heteroreceptor assembly and signaling is poorly understood. We report here a 46-residue, artificial transmembrane protein aptamer, designated ELI-3, that binds and activates the EPOR and induces growth factor independence in murine BaF3 cells expressing the EPOR. ELI-3 requires the transmembrane domain and JAK2-binding sites of the EPOR for activity, but not the cytoplasmic tyrosines that mediate canonical EPOR signaling. Instead, ELI-3-induced proliferation and activation of JAK/STAT signaling requires the transmembrane and cytoplasmic domains of the cytokine receptor β-common subunit (βcR) in addition to the EPOR. Moreover, ELI-3 fails to induce erythroid differentiation of primary human hematopoietic progenitor cells but inhibits nonhematopoietic cell death induced by serum withdrawal., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) can form functional oligomers and in some cases, insoluble disease related aggregates. It is therefore vital to understand processes and mechanisms that control pathway distribution. Divalent cations including Zn 2+ can initiate IDP oligomerisation through the interaction with histidine residues but the mechanisms of doing so are far from understood. Here we apply a multi-disciplinary approach using small angle X-ray scattering, nuclear magnetic resonance spectroscopy, calorimetry and computations to show that that saliva protein Histatin 5 forms highly dynamic oligomers in the presence of Zn 2+ . The process is critically dependent upon interaction between Zn 2+ ions and distinct histidine rich binding motifs which allows for thermodynamic switching between states. We propose a molecular mechanism of oligomerisation, which may be generally applicable to other histidine rich IDPs. Finally, as Histatin 5 is an important saliva component, we suggest that Zn 2+ induced oligomerisation may be crucial for maintaining saliva homeostasis.

The temperature dependence of the conformational properties in simulations of the intrinsically disordered model protein histatin 5 has been investigated using different combinations of force fields, water models, and atomistic and coarse-grained methods. The results have been compared to experimental data obtained from NMR, SAXS, and CD experiments to assess the accuracy and validity of the simulations. The results showed that neither simulations completely agreed with the experimental data, nor did they agree with each other. It was however possible to conclude that the observed conformational changes upon variations in temperature were not at all driven by electrostatic interactions. The final conclusion was that none of the simulations that were investigated in this study was able to accurately capture the temperature induced conformational changes of our model IDP.

β-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer-monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates., Competing Interests: The authors declare no competing financial interest.

Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8-interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8-interacting protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties more consistent with a collapsed pre-molten globule-like conformation, possibly as a consequence of a higher content of π-orbital-containing amino acid residues. Finally, we show that a sizable fraction of ATI2, but not ATI1, is phosphorylated in planta ., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca 2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca 2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca 2+ -bound, Mg 2+ -bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca 2+ and Mg 2+ ) to its EF-hands. At pathologically high Ca 2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties.

Dss1 (also known as Sem1) is a conserved, intrinsically disordered protein with a remarkably broad functional diversity. It is a proteasome subunit but also associates with the BRCA2, RPA, Csn12-Thp1, and TREX-2 complexes. Accordingly, Dss1 functions in protein degradation, DNA repair, transcription, and mRNA export. Here in Schizosaccharomyces pombe, we expand its interactome further to include eIF3, the COP9 signalosome, and the mitotic septins. Within its intrinsically disordered ensemble, Dss1 forms a transiently populated C-terminal helix that dynamically interacts with and shields a central binding region. The helix interfered with the interaction to ATP-citrate lyase but was required for septin binding, and in strains lacking Dss1, ATP-citrate lyase solubility was reduced and septin rings were more persistent. Thus, even weak, transient interactions within Dss1 may dynamically rewire its interactome., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant ( k on ) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity., Competing Interests: Conflict of interest statement: S.G.Y. and Jay D. Horton were both principal investigators on a consortium grant. They did not collaborate directly.

Communication within cells relies on a few protein nodes called hubs, which organize vast interactomes with many partners. Frequently, hub proteins are intrinsically disordered conferring multi-specificity and dynamic communication. Conversely, folded hub proteins may organize networks using disordered partners. In this work, the structure of the RST domain, a unique folded hub, is solved by nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, and its complex with a region of the transcription factor DREB2A is provided through data-driven HADDOCK modeling and mutagenesis analysis. The RST fold is unique, but similar structures are identified in the PAH (paired amphipathic helix), TAFH (TATA-box-associated factor homology), and NCBD (nuclear coactivator binding domain) domains. We designate them as a group the αα hubs, as they share an αα-hairpin super-secondary motif, which serves as an organizing platform for malleable helices of varying topology. This allows for partner adaptation, exclusion, and selection. Our findings provide valuable insights into structural features enabling signaling fidelity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment and computation have been used to study receptor dynamics. Recent studies on three distinct classes of receptors (G-protein coupled receptors, ligand-gated ion-channels and single-pass receptors) are highlighted to show that conformational changes across a range of time-scales and length-scales are central to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial-mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.

NMR spectroscopy has proven to be a key method for studying intrinsically disordered proteins (IDPs). Nonetheless, traditional NMR methods developed for solving structures of ordered protein complexes are insufficient for the full characterization of dynamic IDP complexes, where the energy landscape is broader and more rugged. Furthermore, due to their high sensitivity to environmental changes, NMR studies of IDP complexes must be conducted with extra care and the observed NMR parameters thoroughly evaluated to enable disentanglement of binding events from ensemble distribution changes. In this chapter, written for the non-NMR expert, we start out by outlining sample preparation for IDP complexes, guide through the recording and evaluation of diagnostic 1 H, 15 N-HSQC spectra, and delineate more sophisticated NMR strategies to follow for the particular type of complex. The most relevant experiments are then described in terms of aims, needs, pitfalls, analysis, and expected outcomes, with references to recent examples., (© 2018 Elsevier Inc. All rights reserved.)

Transmembrane domains (TMDs) engage in protein-protein interactions that regulate many cellular processes, but the rules governing the specificity of these interactions are poorly understood. To discover these principles, we analyzed 26-residue model transmembrane proteins consisting exclusively of leucine and isoleucine (called LIL traptamers) that specifically activate the erythropoietin receptor (EPOR) in mouse cells to confer growth factor independence. We discovered that the placement of a single side chain methyl group at specific positions in a traptamer determined whether it associated productively with the TMD of the human EPOR, the mouse EPOR, or both receptors. Association of the traptamers with the EPOR induced EPOR oligomerization in an orientation that stimulated receptor activity. These results highlight the high intrinsic specificity of TMD interactions, demonstrate that a single methyl group can dictate specificity, and define the minimal chemical difference that can modulate the specificity of TMD interactions and the activity of transmembrane proteins.

Intrinsically disordered proteins (IDPs) do not, by themselves, fold into a compact globular structure. They are extremely dynamic and flexible, and are typically involved in signalling and transduction of information through binding to other macromolecules. The reason for their existence may lie in their malleability, which enables them to bind several different partners with high specificity. In addition, their interactions with other macromolecules can be regulated by a variable amount of chemically diverse post-translational modifications. Four kinetically and energetically different types of complexes between an IDP and another macromolecule are reviewed: (1) simple two-state binding involving a single binding site, (2) avidity, (3) allovalency and (4) fuzzy binding; the last three involving more than one site. Finally, a qualitative definition of fuzzy binding is suggested, examples are provided, and its distinction to allovalency and avidity is highlighted and discussed.

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.

Intrinsically disordered proteins (IDPs) are involved in many pivotal cellular processes including phosphorylation and signalling. The structural and functional effects of phosphorylation of IDPs remain poorly understood and difficult to predict. Thus, a need exists to identify motifs that confer phosphorylation-dependent perturbation of the local preferences for forming e.g. helical structures as well as motifs that do not. The disordered distal tail of the Na + /H + exchanger 1 (NHE1) is six-times phosphorylated (S693, S723, S726, S771, T779, S785) by the mitogen activated protein kinase 2 (MAPK1, ERK2). Using NMR spectroscopy, we found that two out of those six phosphorylation sites had a stabilizing effect on transient helices. One of these was further investigated by circular dichroism and NMR spectroscopy as well as by molecular dynamic simulations, which confirmed the stabilizing effect and resulted in the identification of a short linear motif for helix stabilisation: [S/T]-P-{3}-[R/K] where [S/T] is the phosphorylation-site. By analysing IDP and phosphorylation site databases we found that the motif is significantly enriched around known phosphorylation sites, supporting a potential wider-spread role in phosphorylation-mediated regulation of intrinsically disordered proteins. The identification of such motifs is important for understanding the molecular mechanism of cellular signalling, and is crucial for the development of predictors for the structural effect of phosphorylation; a tool of relevance for understanding disease-promoting mutations that for example interfere with signalling for instance through constitutive active and often cancer-promoting signalling., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins play important roles throughout biology, yet our understanding of the relationship between their sequences, structural properties, and functions remains incomplete. The dynamic nature of these proteins, however, makes them difficult to characterize structurally. Many disordered proteins can attain both compact and expanded conformations, and the level of expansion may be regulated and important for function. Experimentally, the level of compaction and shape is often determined either by small-angle x-ray scattering experiments or pulsed-field-gradient NMR diffusion measurements, which provide ensemble-averaged estimates of the radius of gyration and hydrodynamic radius, respectively. Often, these experiments are interpreted using molecular simulations or are used to validate them. We here provide, to our knowledge, a new and efficient method to calculate the hydrodynamic radius of a disordered protein chain from a model of its structural ensemble. In particular, starting from basic concepts in polymer physics, we derive a relationship between the radius of gyration of a structure and its hydrodynamic ratio, which in turn can be used, for example, to compare a simulated ensemble of conformations to NMR diffusion measurements. The relationship may also be valuable when using NMR diffusion measurements to restrain molecular simulations., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains targeting specific cis -acting elements in genes, and by the significant lack of fixed tertiary structure in their extensive intrinsically disordered regions. Recent research in protein intrinsic disorder (ID) has changed our understanding of transcriptional activation domains from 'negative noodles' to ID regions with function-related, short sequence motifs and molecular recognition features with structural propensities. This review focuses on molecular aspects of TFs, which represent paradigms of ID-related features. Through specific examples, we review how the ID-associated flexibility of TFs enables them to participate in large interactomes, how they use only a few hydrophobic residues, short sequence motifs, prestructured motifs, and coupled folding and binding for their interactions with co-activators, and how their accessibility to post-translational modification affects their interactions. It is furthermore emphasized how classic biochemical concepts like allostery, conformational selection, induced fit, and feedback regulation are undergoing a revival with the appreciation of ID. The review also describes the most recent advances based on computational simulations of ID-based interaction mechanisms and structural analysis of ID in the context of full-length TFs and suggests future directions for research in TF ID., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4 Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4 Cdt2 , by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside transporter human equilibrative nucleoside transporter 1 (hENT1) and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4 Cdt2 inactivation, suggesting that Spd1-in addition to repressing dNTP synthesis-together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.

Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions. These types of analyses determine the amounts and proportions of individual constituents that participate in a reaction as well as their rates of reactions and their thermodynamics. Although many different methods are available, there is currently no single method able to quantitatively capture and describe all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates, and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative descriptions of protein interactions. In this review we provide an easy-access approach to NMR for the non-NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background and illustrate simple protein-ligand interactions as well as typical strategies for measuring binding constants using NMR spectroscopy. Finally, this review provides examples of caveats of the method as well as the options to improve the outcome of an NMR analysis of a protein interaction reaction., (Published by Wiley-Blackwell. © 2016 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

The human immunodeficiency virus-1 (HIV-1) protease is a complex protein that in its active form adopts a homodimer dominated by β-sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1 protease that is populated above 0 °C and therefore directly accessible to various spectroscopic approaches. Using nuclear magnetic resonance secondary chemical shifts, temperature coefficients, and protein dynamics, we suggest that the cold-denatured state populates a compact wet globule containing transient non-native-like α-helical elements. From the linearity of the temperature coefficients and the hydrodynamic radii, we propose that the overall architecture of the cold-denatured state is maintained over the temperature range studied.

Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein radical-induced cell death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound by RCD1, we analyzed the IDRs in three protein partners, DREB2A (dehydration-responsive element-binding protein 2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as (DE)X(1,2)(YF)X(1,4)(DE)L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher K d values. Specifically, although the SLiM was surrounded by IDRs, individual intrinsic α-helix propensities varied as shown by CD spectroscopy. NMR spectroscopy further demonstrated that DREB2A underwent coupled folding and binding with α-helix formation upon interaction with RCD1, whereas peptides from ANAC013 and ANAC046 formed different structures or were fuzzy in the complexes. These findings allow us to present a model of the stress-associated RCD1-transcription factor interactome and to contribute to the emerging understanding of the interactions between folded hubs and their intrinsically disordered partners., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Single-pass transmembrane receptors are involved in essential processes of both physiological and pathological nature and represent more than 1300 proteins in the human genome. Despite the high biological relevance of these receptors, the mechanisms of the signal transductions they facilitate are incompletely understood. One major obstacle is the lack of structures of the transmembrane domains that connect the extracellular ligand-binding domains to the intracellular signaling platforms. Over a period of almost 20 years since the first structure was reported, only 21 of these receptors have become represented by a transmembrane domain structure. This scarceness stands in strong contrast to the significance of these transmembrane α-helices for receptor functionality. In this review, we explore the properties and qualities of the current set of structures, as well as the methodological difficulties associated with their characterization and the challenges left to be overcome. Without an increased and focused effort to bring this class of proteins on par with the remaining membrane protein field, a serious lag in their biological understanding looms. Design of pharmaceutical agents, prediction of mutational affects in relation to disease, and deciphering of functional mechanisms require high-resolution structural information, especially when dealing with a domain carrying so much functionality in so few residues., (© 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Prolactin (PRL) and its receptor (PRLR) are implicated in breast cancer invasiveness, although their exact roles remain controversial. The Na(+)/H(+) exchanger (NHE1) plays essential roles in cancer cell motility and invasiveness, but the PRLR and NHE1 have not previously been linked. Here we show that in T47D human breast cancer cells, which express high levels of PRLR and NHE1, exposure to PRL led to the activation of Janus kinase-2 (JAK2)/signal transducer and activator of transcription-5 (STAT5), Akt, and ERK1/2 signaling and the rapid formation of peripheral membrane ruffles, known to be associated with cell motility. NHE1 was present in small ruffles prior to PRL treatment and was further recruited to the larger, more dynamic ruffles induced by PRL exposure. In PRL-induced ruffles, NHE1 colocalized with activated Akt, ERK1/2, and the ERK effector p90Ribosomal S kinase (p90RSK), known regulators of NHE1 activity. Stimulation of T47D cells with PRL augmented p90RSK activation, Ser703-phosphorylation of NHE1, NHE1-dependent intracellular pH recovery, pericellular acidification, and NHE1-dependent invasiveness. NHE1 activity and localization to ruffles were attenuated by the inhibition of Akt and/or ERK1/2. In contrast, noncancerous MCF10A breast epithelial cells expressed NHE1 and PRLR at lower levels than T47D cells, and their stimulation with PRL induced neither NHE1 activation nor NHE1-dependent invasiveness. In conclusion, we show for the first time that PRLR activation stimulates breast cancer cell invasiveness via the activation of NHE1. We propose that PRL-induced NHE1 activation and the resulting NHE1-dependent invasiveness may contribute to the metastatic behavior of human breast cancer cells.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.

The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

DSS1/Sem1 is a versatile intrinsically disordered protein. Besides being a bona fide subunit of the 26S proteasome, DSS1 associates with other protein complexes, including BRCA2-RPA, involved in homologous recombination; the Csn12-Thp3 complex, involved in RNA splicing; the integrator, involved in transcription; and the TREX-2 complex, involved in nuclear export of mRNA and transcription elongation. As a subunit of the proteasome, DSS1 functions both in complex assembly and possibly as a ubiquitin receptor. Here, we summarise structural and functional aspects of DSS1/Sem1 with particular emphasis on its multifunctional and disordered properties. We suggest that DSS1/Sem1 can act as a polyanionic adhesive to prevent nonproductive interactions during construction of protein assemblies, uniquely employing different structures when associating with the diverse multisubunit complexes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding., Methods and Results: Here, we identify the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner., Conclusions: This work characterizes a new type of scaffolding complex, which we term a "shuffle complex", between the disordered hNHE1-tail and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2.

Non-homologous end-joining (NHEJ) is an essential DNA double strand break repair pathway during all cell cycle stages. Deficiency in NHEJ factors can lead to accumulation of unrepaired DNA breaks or faulty DNA repair, which may ultimately result in cell death, senescence or carcinogenesis. The Ku70/80 heterodimer is a key-player in the NHEJ pathway and binds to DNA termini with high affinity, where it helps to protect DNA ends from degradation and to recruit other NHEJ factors required for repair. The mechanism of Ku70/80 detachment from the DNA helix after completion of DNA repair is incompletely understood. Some data suggest that certain DNA repair factors are ubiquitylated and targeted for proteasomal degradation after repair. Recent studies suggest that Ku80 is conjugated to lysine48-linked ubiquitin chains by the Skp1-Cullin-F-box (SCF) complex and/or the RING finger protein 8 (RNF8) ubiquitin-protein ligases, followed by rapid proteasomal degradation. In this review we address the structure and function of the Ku70/80 heterodimer and how ubiquitylation may affect the release of Ku70/80 from chromatin and its subsequent degradation via the ubiquitin-proteasome system.

Despite the biological and pharmaceutical significance of membrane proteins, their tertiary structures constitute less than 3% of known structures. One of the major obstacles for initiating structural studies of membrane proteins by NMR spectroscopy is the generation of high amounts of isotope-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures of single-pass TMDs, which are difficult to solve by other means.

De novo design and chemical synthesis of proteins and of other artificial structures that mimic them is a central strategy for understanding protein folding and for accessing proteins with new functions. We have previously described carbohydrates that act as templates for the assembly of artificial proteins, so-called carboproteins. The hypothesis is that the template preorganizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from the template could facilitate protein folding. Here we report the design and synthesis of three-helix-bundle carboproteins on deoxyhexopyranosides. The carboproteins were analyzed by CD, analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and NMR spectroscopy, and this revealed the formation of the first compact and folded monomeric carboprotein, distinctly different from a molten globule. En route to this carboprotein we observed a clear effect originating from the template on protein folding., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In biology proteins from different structural classes interact across and within classes in ways that are optimized to achieve balanced functional outputs. The interactions between intrinsically disordered proteins (IDPs) and other proteins rely on changes in flexibility and this is seen as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol(-1).