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Showing papers on "Structural biology published in 2016"


Journal ArticleDOI
TL;DR: PEP-FOLD3 is a novel computational framework that allows both (i) de novo free or biased prediction for linear peptides between 5 and 50 amino acids, and (ii) the generation of native-like conformations of peptides interacting with a protein when the interaction site is known in advance.
Abstract: Structure determination of linear peptides of 5-50 amino acids in aqueous solution and interacting with proteins is a key aspect in structural biology. PEP-FOLD3 is a novel computational framework, that allows both (i) de novo free or biased prediction for linear peptides between 5 and 50 amino acids, and (ii) the generation of native-like conformations of peptides interacting with a protein when the interaction site is known in advance. PEP-FOLD3 is fast, and usually returns solutions in a few minutes. Testing PEP-FOLD3 on 56 peptides in aqueous solution led to experimental-like conformations for 80% of the targets. Using a benchmark of 61 peptide-protein targets starting from the unbound form of the protein receptor, PEP-FOLD3 was able to generate peptide poses deviating on average by 3.3A from the experimental conformation and return a native-like pose in the first 10 clusters for 52% of the targets. PEP-FOLD3 is available at http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3.

627 citations


Journal ArticleDOI
02 Feb 2016-eLife
TL;DR: It is shown that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA), which are found in canonical nucleolar localization signals.
Abstract: Inside cells, machines called ribosomes assemble proteins from building blocks known as amino acids. Cells can alter the numbers of ribosomes they produce to match the cell’s demand for new proteins. For instance, when cells grow they require a lot of new proteins and therefore more ribosomes are produced. However, when cells face harsh conditions that cause stress (e.g. exposure to UV radiation or a harmful chemical) they generally stop growing and therefore need fewer ribosomes. In human and other eukaryotic cells, ribosomes are assembled in a structure called the nucleolus. However, because the nucleolus is not separated from the rest of the cell by a membrane, it was not clear how it is able to accumulate large quantities of the proteins and other molecules needed to make ribosomes. Recent work suggests that the nucleolus is formed through a process referred to as “phase separation” in which the liquid in a particular region of the cell has different physical properties to the liquid surrounding it. This is like how oil and water form separate layers when mixed. A protein called nucleophosmin is found at high levels in the nucleolus where it interacts with many other proteins, including those involved in making ribosomes. Nucleophosmin binds to motifs within these proteins that contain multiple copies of an amino acid called arginine (referred to as R-motifs). Now, Mitrea et al. investigate how nucleophosmin binds to R-motif proteins and whether this is important for assembling the nucleolus. A search for R-motifs in a list of over a hundred proteins known to bind to nucleophosmin showed that the majority of these proteins contained multiple R-motifs. Furthermore, when high levels of nucleophosmin and the R-motif proteins were present, they underwent phase separation. Next, Mitrea et al. examine the changes in how nucleophosmin and a ribosomal protein interact before and after phase separation. The experiments show that many molecules of nucleophosmin bind to each other and that multiple regions in nucleophosmin are able to interact with the R-motifs. Together, these interactions produce large assemblies of proteins that result in the creation of separate liquid layers. Furthermore, the experiments show that R-motif proteins and other molecules needed to make ribosomes can be brought together within the same liquid phase by nucleophosmin. Mitrea et al.’s findings provide the first insights into the role of nucleophosmin in the molecular organisation of the nucleolus. The next challenge is to understand how this organisation promotes the production of ribosomes and helps the cell to respond to stressful situations.

374 citations


Book
01 Jan 2016
TL;DR: This work describes the development and application of the RISM-SCF/MCSCF approach for the chemical processes in solutions nucleic acids simulations membrane simulations, and the design and design of computer-aided drug design.
Abstract: Computational methods: atomistic models and force fields dynamics methods conformational analysis treatment of long-range forces and potential internal co-ordinate simulation method implicit solvent models normal mode analysis of biological molecules free energy calculations reaction rates and transition pathways computer simulation of biochemical reactions with QM-MM methods Experimental data analysis: X-ray and neutron scattering as probes of the dynamics of biological molecules applications of molecular modelling in NMR structure determination Modelling and design: comparative protein structure modelling Bayesian statistics in molecular and structural biology Computer-aided drug design Advanced applications: protein folding simulations of electron transfer proteins the RISM-SCF/MCSCF approach for the chemical processes in solutions nucleic acids simulations membrane simulations Appendix: useful Web sites

364 citations


Journal ArticleDOI
14 Sep 2016-eLife
TL;DR: The crystal structure of the catalytic core of the human m6A writer complex comprising METTL3 and METTL14 is reported and the heterodimeric architecture of the complex and donor substrate binding byMETTL3 is revealed.
Abstract: Methylation of adenosines at the N(6) position (m(6)A) is a dynamic and abundant epitranscriptomic mark that regulates critical aspects of eukaryotic RNA metabolism in numerous biological processes. The RNA methyltransferases METTL3 and METTL14 are components of a multisubunit m(6)A writer complex whose enzymatic activity is substantially higher than the activities of METTL3 or METTL14 alone. The molecular mechanism underpinning this synergistic effect is poorly understood. Here we report the crystal structure of the catalytic core of the human m(6)A writer complex comprising METTL3 and METTL14. The structure reveals the heterodimeric architecture of the complex and donor substrate binding by METTL3. Structure-guided mutagenesis indicates that METTL3 is the catalytic subunit of the complex, whereas METTL14 has a degenerate active site and plays non-catalytic roles in maintaining complex integrity and substrate RNA binding. These studies illuminate the molecular mechanism and evolutionary history of eukaryotic m(6)A modification in post-transcriptional genome regulation.

344 citations


Journal ArticleDOI
15 Sep 2016-Nature
TL;DR: Developments in the electron microscopy of frozen hydrated samples (cryo-electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules, resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology.
Abstract: Knowledge of the three-dimensional structures of proteins and other biological macromolecules often aids understanding of how they perform complicated tasks in the cell. Because many such tasks involve the cleavage or formation of chemical bonds, structural characterization at the atomic level is most useful. Developments in the electron microscopy of frozen hydrated samples (cryo-electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules. This is resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology.

341 citations


Journal ArticleDOI
TL;DR: Nanodisc technology provides important advantages for the isolation, purification, structural resolution and functional characterization of membrane proteins, and the ability to precisely control the nanodisc composition provides a nanoscale membrane surface for investigating molecular recognition events.
Abstract: Membrane proteins have long presented a challenge to biochemical and functional studies. In the absence of a bilayer environment, individual proteins and critical macromolecular complexes may be insoluble and may display altered or absent activities. Nanodisc technology provides important advantages for the isolation, purification, structural resolution and functional characterization of membrane proteins. In addition, the ability to precisely control the nanodisc composition provides a nanoscale membrane surface for investigating molecular recognition events.

336 citations


Journal ArticleDOI
TL;DR: Polymer concepts provide an important basis for relating the physical properties of unstructured proteins to folding and function and a reemergence of polymer physics as a versatile framework for understanding their structure and dynamics is seen.
Abstract: The properties of unfolded proteins have long been of interest because of their importance to the protein folding process. Recently, the surprising prevalence of unstructured regions or entirely disordered proteins under physiological conditions has led to the realization that such intrinsically disordered proteins can be functional even in the absence of a folded structure. However, owing to their broad conformational distributions, many of the properties of unstructured proteins are difficult to describe with the established concepts of structural biology. We have thus seen a reemergence of polymer physics as a versatile framework for understanding their structure and dynamics. An important driving force for these developments has been single-molecule spectroscopy, as it allows structural heterogeneity, intramolecular distance distributions, and dynamics to be quantified over a wide range of timescales and solution conditions. Polymer concepts provide an important basis for relating the physical properties of unstructured proteins to folding and function.

263 citations


Journal ArticleDOI
01 Nov 2016-eLife
TL;DR: Protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening, and metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects.
Abstract: Much of the work that has been done to understand how cells work has involved studying parts of a cell in isolation This is particularly true of studies that have examined the arrangement of atoms in large molecules with elaborate structures like proteins or DNA However, cells are densely packed with many different molecules and there is little proof that proteins keep the same structures inside cells that they have when they are studied alone To really understand how cells work, new ways to understand how molecules behave inside cells are needed While this cannot be achieved directly, technology has now reached the stage where we can, to some extent, study living cells by recreating them virtually Simulated cells can copy the atomic details of all the molecules in a cell and can estimate how different molecules might behave together Yu et al have now developed a computer simulation of part of a cell from the bacterium, Mycoplasma genitalium, one of the simplest forms of life on Earth This model suggested new possible interactions between molecules inside cells that cannot currently be studied in real cells The model shows that some proteins have a much less rigid structure in cells than they do in isolation, whilst others are able to work together more closely to carry out certain tasks Finally, the model predicted that small molecules such as food, water and drugs would move more slowly through cells as they become stuck or trapped by larger molecules These results could be particularly important in helping to improve drug design Currently the simulations are limited, and can only model parts of simple cells for less than a thousandth of a second However, in future it should be possible to recreate larger and more complex cells, including human cells, for longer periods of time These could be used to better study human diseases and help to design new treatments The ultimate goal is to simulate a whole cell in full detail by combining all the available experimental data

248 citations


Journal ArticleDOI
TL;DR: The structural basis of the principal nuclear import pathways and the molecular basis of their regulation are reviewed and post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

195 citations


Journal ArticleDOI
TL;DR: Recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins agree well with those identified by complementary experimental techniques, demonstrating the power of the molecular dynamics approach in the prediction and characterization of lipid interaction Sites on integral membrane proteins.

147 citations


Journal ArticleDOI
TL;DR: This study presents the structure of IDO1 in complex with 24, a NLG919 analogue with potent activity and demonstrates that extensive hydrophobic interactions and the unique hydrogen bonding network contribute to the great potency of imidazoleisoindole derivatives.
Abstract: Indoleamine 2,3-dioxygenase 1 (IDO1), promoting immune escape of tumors, is a therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have been identified, but only limited structural biology studies of IDO1 inhibitors are available to provide insights on the binding mechanism of IDO1. In this study, we present the structure of IDO1 in complex with 24, a NLG919 analogue with potent activity. The complex structure revealed the imidazole nitrogen atom of 24 to coordinate with the heme iron, and the imidazoleisoindole core situated in pocket A with the 1-cyclohexylethanol moiety extended to pocket B to interact with the surrounding residues. Most interestingly, 24 formed an extensive hydrogen bond network with IDO1, which is a distinct feature of IDO1/24 complex structure and is not observed in the other IDO1 complex structures. Further structure–activity relationship, UV spectra, and structural biology studies of several analogues of 24 demonstrated that extensive hydrophobic interactio...

Journal ArticleDOI
TL;DR: Studying structure can reveal how molecules have evolved, and this type of insight would otherwise be lost by looking at only the molecule’s sequence.

Journal ArticleDOI
TL;DR: A combination of ion‐mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase and showed that for low charge states under gentle conditions, aspects of the native secondary and secondary structure can be conserved.
Abstract: Can the structures of small to medium-sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion-mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β-lactoglobulin, which are prototypical examples of helical and β-sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved.

Journal ArticleDOI
TL;DR: Hepatitis B virus is a virion where the envelope proteins have multiple structures, the envelope-capsid interaction is irregular, and the capsid is a dynamic compartment that actively participates in metabolism of the encapsidated genome and carries regulated signals for intracellular trafficking.
Abstract: Hepatitis B virus is one of the smallest human pathogens, encoded by a 3,200-bp genome with only four open reading frames. Yet the virus shows a remarkable diversity in structural features, often with the same proteins adopting several conformations. In part, this is the parsimony of viruses, where a minimal number of proteins perform a wide variety of functions. However, a more important theme is that weak interactions between components as well as components with multiple conformations that have similar stabilities lead to a highly dynamic system. In hepatitis B virus, this is manifested as a virion where the envelope proteins have multiple structures, the envelope-capsid interaction is irregular, and the capsid is a dynamic compartment that actively participates in metabolism of the encapsidated genome and carries regulated signals for intracellular trafficking.

Journal ArticleDOI
TL;DR: The model emerging from the data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein.
Abstract: Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered multidrug transporter LmrP from Lactococcus lactis, using the pattern of distances between spin-label pairs previously shown to report on alternating access of the protein. We uncovered, at the molecular level, how the lipid headgroups shape the conformational-energy landscape of the transporter. The model emerging from our data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers.

Journal ArticleDOI
TL;DR: The characterisation of the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds.
Abstract: The hydrophobic effect is a major driving force in protein folding. A complete understanding of this effect requires the description of the conformational states of water and protein molecules at different temperatures. Towards this goal, we characterise the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations. A detailed analysis of the resulting structures reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds. Thus, even if proteins are ‘large’ particles (in terms of the hydrophobic effect, i.e. larger than 1 nm), because of the presence of complex surface patterns of polar and non-polar residues their behaviour can be compared to that of ‘small’ particles (i.e. smaller than 1 nm). We thus find that the hot denatured state is more compact and richer in secondary structure than the cold denatured state, since water at lower temperatures can form more hydrogen bonds than at high temperatures. Then, using Φ-value analysis we show that the structural differences between the hot and cold denatured states result in two alternative folding mechanisms. These findings thus illustrate how the analysis of water-protein hydrogen bonds can reveal the molecular origins of protein behaviours associated with the hydrophobic effect.

Journal ArticleDOI
TL;DR: This review focuses on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy (cryo-EM).
Abstract: Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Journal ArticleDOI
TL;DR: A thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters and facilitated structure-based drug design applied to GPCRs.
Abstract: The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

Journal ArticleDOI
TL;DR: The power of nuclear magnetic resonance spectroscopy is highlighted to untangle complex relationships behind molecular chaperones and their mechanism(s) of action.

Journal ArticleDOI
20 May 2016-eLife
TL;DR: It is shown that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities.
Abstract: Animal, plant and fungal cells contain a structure called the nucleus, inside which the genetic material of the cell is stored. For the cell to work properly, certain proteins and other molecules need to be able to enter and exit the nucleus. This transport is carried out by pore-like molecular “devices” known as Nuclear Pore Complexes, whose architecture and mode of operation are unique among cellular transporters. Nuclear Pore Complexes are charged with a daunting task of deciding which of the hundreds of molecules it conducts per second should go through and which should not. Small molecules can pass freely through Nuclear Pore Complexes. However, larger molecules can only pass through the pore efficiently if they are bound to specialized transport proteins that interact with the proteins – called FG nucleoporins – that line the pore. A unique feature of the FG nucleoporins is that, unlike typical proteins, they do not have a defined three-dimensional structure. Instead, they form a soft and pliable lining inside the Nuclear Pore Complex passageway. Exactly how interacting with transport proteins affects the structure and spatial arrangements of the FG nucleoporins in a way that allows them to control transport is not well understood. This is in part because existing experimental techniques are unable to study the structures of the FG nucleoporins in enough detail to track how they change during transport. The complexity and the diversity of the FG nucleoporins also make them difficult to model in detail. Vovk, Gu et al. have developed a theoretical model that is based on just three basic physical properties of the FG nucleoporins – their flexibility, their ability to interact with each other, and their binding with the transport proteins. Future work can refine the model by incorporating further molecular details about the interactions between FG nucleoporins and transport proteins. The predictions made by this simple model agree well with experimental results in a wide range of situations – from single molecules to complex spatial assemblies. They also explain why some of the experimental results appear to contradict each other and suggest how several outstanding controversies in the field can be reconciled. Because the model invokes only fundamental physical principles of FG nucleoporin assemblies, it shows that some of their general properties do not depend on the exact conditions. In particular, this might shed light on why Nuclear Pore Complexes in different organisms perform essentially the same function, although the details of their molecular structure may differ. This also suggests how the FG nucleoporins can be manipulated to build artificial devices based on the same principles.

Book ChapterDOI
TL;DR: The structures of NRPS proteins are described and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains.
Abstract: The nonribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains.

Journal ArticleDOI
TL;DR: In-cell nuclear magnetic resonance can be applied to several cellular systems to obtain biologically relevant structural and functional information and focus on the applications to protein folding, interactions, and post-translational modifications.

Journal ArticleDOI
27 Oct 2016-eLife
TL;DR: A crystallographic structure of the minimal adhesive fragment of the zebrafish PCDh19 extracellular domain reveals the adhesive interface for Pcdh19, which is broadly relevant to both non-clustered δ and clustered protocadherin subfamilies, and reveals the biochemical basis of their pathogenic effects during brain development.
Abstract: As the brain develops, its basic building blocks – cells called neurons – need to form the correct connections with one another in order to give rise to neural circuits A mistake that leads to the formation of incorrect connections can result in a number of disorders or brain abnormalities Proteins called cadherins that are present on the surface of neurons enable them to stick to their correct partners like Velcro One of these proteins is called Protocadherin-19 However, it was not fully understood how this protein forms an adhesive bond with other Protocadherin-19 molecules, or how some of the proteins within the cadherin family are able to distinguish between one another Cooper et al used X-ray crystallography to visualize the molecular structure of Protocadherin-19 taken from zebrafish in order to better understand the adhesive bond that these proteins form with each other In addition, the new structure showed the sites of the mutations that cause a form of epilepsy in infant females From this, Cooper et al could predict how the mutations would disrupt Protocadherin-19’s shape and function The structures revealed that Protocadherin-19 molecules from adjacent cells engage in a “forearm handshake” to form the bond that connects neurons Some of the mutations that cause epilepsy occur in the region responsible for this Protocadherin-19 forearm handshake Laboratory experiments confirmed that these mutations impair the formation of the adhesive bond, revealing the molecular basis for some of the mutations that underlie Protocadherin-19-female-limited epilepsy Other cadherin molecules may interact via a similar forearm handshake; this could be investigated in future experiments It also remains to be discovered how brain wiring depends on Protocadherin-19 adhesion in animal development, and how altering these proteins can rewire developing brain circuits

Journal ArticleDOI
TL;DR: High-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein.
Abstract: Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein. Activins are members of the TGF-β family of growth factors that are processed from precursors into the mature proteins. Here, the authors use structural biology and biochemistry to examine the protein domain organisation and gain insights into the activation of pro-activin A.

Journal ArticleDOI
09 Nov 2016-eLife
TL;DR: Using Nuclear Magnetic Resonance spectroscopy, this work reveals how disease-causing mutations in p97 deregulate dynamics of the N-terminal domain that binds adaptor proteins involved in controlling p97 function, providing a molecular basis for understanding how malfunction occurs.
Abstract: p97/VCP is an essential, abundant AAA+ ATPase that is conserved throughout eukaryotes, with central functions in diverse processes ranging from protein degradation to DNA damage repair and membrane fusion. p97 has been implicated in the etiology of degenerative diseases and in cancer. Using Nuclear Magnetic Resonance spectroscopy we reveal how disease-causing mutations in p97 deregulate dynamics of the N-terminal domain that binds adaptor proteins involved in controlling p97 function. Our results provide a molecular basis for understanding how malfunction occurs whereby mutations shift the ADP-bound form of the enzyme towards an ATP-like state in a manner that correlates with disease severity. This deregulation interferes with the two-pronged binding of an adaptor that affects p97 function in lysosomal degradation of substrates. Subtle structural changes propagate from mutation sites to regions distal in space, defining allosteric networks that facilitate inter-domain communication, with potential implications for modulation of enzyme activity by drug molecules.

Journal ArticleDOI
03 Dec 2016-eLife
TL;DR: This work combines sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution and finds that TatA also associates with TatC at the polar cluster site.
Abstract: The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

Journal ArticleDOI
TL;DR: It is shown that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility.
Abstract: Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function. Mutations in aggregation prone regions of recombinant proteins often improve their solubility, although they might cause negative effects on their structure and function. Here, the authors identify proteins hot spots that can be exploited to optimize solubility without compromising stability.

Journal ArticleDOI
TL;DR: This work illustrates how structural free energy landscapes and fitness landscapes of proteins can be used in an integrated way, and in the context of kinase family proteins, can potentially impact therapeutic design strategies.
Abstract: Understanding the conformational propensities of proteins is key to solving many problems in structural biology and biophysics. The co-variation of pairs of mutations contained in multiple sequence alignments of protein families can be used to build a Potts Hamiltonian model of the sequence patterns which accurately predicts structural contacts. This observation paves the way to develop deeper connections between evolutionary fitness landscapes of entire protein families and the corresponding free energy landscapes which determine the conformational propensities of individual proteins. Using statistical energies determined from the Potts model and an alignment of 2896 PDB structures, we predict the propensity for particular kinase family proteins to assume a "DFG-out" conformation implicated in the susceptibility of some kinases to type-II inhibitors, and validate the predictions by comparison with the observed structural propensities of the corresponding proteins and experimental binding affinity data. We decompose the statistical energies to investigate which interactions contribute the most to the conformational preference for particular sequences and the corresponding proteins. We find that interactions involving the activation loop and the C-helix and HRD motif are primarily responsible for stabilizing the DFG-in state. This work illustrates how structural free energy landscapes and fitness landscapes of proteins can be used in an integrated way, and in the context of kinase family proteins, can potentially impact therapeutic design strategies.

Journal ArticleDOI
26 Oct 2016-eLife
TL;DR: The wider mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.
Abstract: Transcription factors are proteins that can bind to DNA to regulate the activity of genes One family of transcription factors in mammals is known as the bHLH-PAS family, which consists of sixteen members including NPAS1 and NPAS3 These two proteins are both found in nerve cells, and genetic mutations that affect NPAS1 or NPAS3 have been linked to psychiatric conditions in humans Therefore, researchers would like to discover new drugs that can bind to these proteins and control their activities in nerve cells Understanding the three-dimensional structure of a protein can aid the discovery of small molecules that can bind to these proteins and act as drugs Proteins in the bHLH-PAS family have to form pairs in order to bind to DNA: NPAS1 and NPAS3 both interact with another bHLH-PAS protein called ARNT, but it is not clear exactly how this works In 2015, a team of researchers described the shapes that ARNT adopts when it forms pairs with two other bHLH-PAS proteins that are important for sensing when oxygen levels drop in cells Here, Wu et al – including many of the researchers involved in the earlier work – have used a technique called X-ray crystallography to determine the three-dimensional shapes of NPAS1 when it is bound to ARNT, and NPAS3 when it is bound to both ARNT and DNA The experiments show that each of these structures contains four distinct pockets that certain small molecules might be able to bind to The NPAS1 and NPAS3 structures are similar to each other and to the previously discovered bHLH-PAS structures involved in oxygen sensing Further analysis of other bHLH-PAS proteins suggests that all the members of this protein family are likely to be able to bind to small molecules and should therefore be considered as potential drug targets The next step following on from this work is to identify small molecules that bind to bHLH-PAS proteins, which will help to reveal the genes that are regulated by this family In the future, these small molecules may have the potential to be developed into new drugs to treat psychiatric conditions and other diseases in humans

Journal ArticleDOI
TL;DR: It is demonstrated that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones and sequence variability predicted from TERM data agrees closely with evolutionary variation.
Abstract: Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence-a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure.