Ensemble Of Proteins Research Articles

Synergy and allostery in ligand binding by HIV-1 Nef.

The Nef protein of human and simian immunodeficiency viruses boosts viral pathogenicity through its interactions with host cell proteins. By combining the polyvalency of its large unstructured regions with the binding selectivity and strength of its folded core domain, Nef can associate with many different host cell proteins, thereby disrupting their functions. For example, the combination of a linear proline-rich motif and hydrophobic core domain surface allows Nef to bind tightly and specifically to SH3 domains of Src family kinases. We investigated whether the interplay between Nef's flexible regions and its core domain could allosterically influence ligand selection. We found that the flexible regions can associate with the core domain in different ways, producing distinct conformational states that alter the way in which Nef selects for SH3 domains and exposes some of its binding motifs. The ensuing crosstalk between ligands might promote functionally coherent Nef-bound protein ensembles by synergizing certain subsets of ligands while excluding others. We also combined proteomic and bioinformatics analyses to identify human proteins that select SH3 domains in the same way as Nef. We found that only 3% of clones from a whole-human fetal library displayed Nef-like SH3 selectivity. However, in most cases, this selectivity appears to be achieved by a canonical linear interaction rather than by a Nef-like ‘tertiary' interaction. Our analysis supports the contention that Nef's mode of hijacking SH3 domains is a virus-specific adaptation with no or very few cellular counterparts. Thus, the Nef tertiary binding surface is a promising virus-specific drug target.

Multifunctionality and intrinsic disorder of royal jelly proteome.

Royal Jelly (RJ) is a gelatinous white-yellowish fluid, possessing a sour taste and a slight phenolic smell that is secreted by the hypopharyngeal and mandibular salivary glands of the nurse honeybees, and is used in nutrition of larvae and adult queens. Similar to other substances associated with the activities of honeybees, RJ not only contains nutritive components, such as carbohydrates, proteins, peptides, lipids, vitamins, and mineral salts,but also represents a natural ingredient with cosmetic and health-promoting properties. RJ is characterized by remarkable multifunctionality, possessing numerous biological activities. Although this multifunctionality of RJ can be considered as a consequence of its complex nature, many proteins and peptides in RJ are polyfunctional entities themselves. In this article, we show that RJ proteins contain different levels of intrinsic disorder, have sites of post-translational modifications, can be found in multiple isoforms, and many of them possess disorder-based binding sites, suggesting that the conformational ensembles of the RJ proteins might undergo change as a result of their interaction with specific binding partners. All these observations suggest that the multifunctionality of proteins and peptides from RJ is determined by their structural heterogeneity and polymorphism, and serve as an illustration of the protein structure-function continuum concept.

Generating Protein Ensembles by Applying Forces to an Improved Elastic Network Model

The lack of experimental structures showing sufficient conformational diversity introduces limitations for the study of protein mechanisms. Computed ensembles of protein conformations can aid in investigations of functional mechanisms. Application of our recent development of a protein packing based hinge prediction method (PACKMAN) and an improved elastic network model to study protein global motions (hd-ANM) together enable improved protein ensemble generations. Here, we apply random forces on the surface to generate the ensemble. Multiple independent Monte Carlo Markov Chain force applications and energy based sampling is implemented to obtain a protein ensemble. We observe a 70% PCA overlap between an ensemble generated by using our method with that of the corresponding protein crystal structure ensemble available in the PDB database. Moreover, results provide mechanistic insights into the conformational transition pathway of proteins. We aim to utilize native and mutant protein ensembles generated using our method to estimate the change in global motion of protein and the allosteric pathway changes induced by a point mutation.

Integrating NMR, SAXS and Single-Molecule FRET Data to Infer Conformational Ensembles of the Yeast Sic1 Protein

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with non-smFRET (NMR) data and reserved the smFRET data as an independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. The results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.

Sequence and Chemical Environment Determine the Global Dimension of Intrinsically Disordered Protein Ensembles

Intrinsically disordered proteins (IDPs) play essential roles in the cell, but do not have a stable native structure. Lacking intramolecular bonds, IDPs exist in a rapidly changing conformational ensemble. These ensembles have a high degree of surface area exposure, making them sensitive to the physical-chemical composition of the cellular environment. The sensitivity of IDPs to their environment can lead to a solution-induced change in both global ensemble dimensions (such as radius of gyration) and local residual structure between residues, which may alter IDP function. To reveal the link between IDP sequence and the sensitivity of its ensemble to physical-chemical changes in its environment we use implicit-solvent Monte Carlo simulations of IDP ensembles in a range of solution conditions using a method we call Solution Space Scanning. We use Solution Space Scanning to compile a dataset containing over 100 naturally occurring IDPs in different solution conditions. Our dataset reveals a staggering range of IDP sensitivities, which shows a weak correlation with sequence length and chain-average molecular features such as hydrophobicity or net charge. Instead, we find a strong correlation between IDPs solution sensitivity and the global dimension of the chain in aqueous buffer. We corroborate our findings with high-throughput experiments and a polymer physics model to establish how sensitivity to solution conditions emerges in intrinsically disordered proteins.

Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.

Cu2+, Ca2+, and methionine oxidation expose the hydrophobic α-synuclein NAC domain

α-Synuclein is an intrinsically disordered protein whose aggregation is related to Parkinson's disease and other neurodegenerative disorders. Metal cations are one of the main factors affecting the propensity of α-synuclein to aggregate, either by directly binding to it or by catalyzing the production of reactive oxygen species that oxidize it. His50, Asp121 and several additional C-terminal α-synuclein residues are binding sites for numerous metal cations, while methionine sulfoxidation occurs readily on this protein under oxidative stress conditions. Molecular dynamics simulations are an excellent tool to obtain a microscopic picture of how metal binding or methionine sulfoxidation alter the conformational preferences of α-synuclein and, hence, its aggregation propensity. In this work, we report the first coarse-grained molecular dynamics study comparing the conformational ensembles of the native protein, the protein bound to either Cu2+ or Ca2+ at its main binding sites, and the methionine-sulfoxidized protein. Our results suggest that these events alter the transient α-synuclein intramolecular contacts, inducing a greater solvent exposure of its hydrophobic, aggregation-prone NAC domain, in full agreement with a recent experimental study on Ca2+ binding. Moreover, metal-binding residues directly participate in the long-range contacts that shield this domain and regulate α-synuclein aggregation. These results provide a molecular-level rationalization of the enhanced fibrillation experimentally observed in the presence of Cu2+ or Ca2+ and the oligomerization induced by methionine sulfoxidation.

Silver in biology and medicine: opportunities for metallomics researchers.

The antibacterial properties of silver have been known for centuries and the threat of antibiotic-resistant bacteria has led to renewed focus on the noble metal. Silver is now commonly included in a range of household and medical items to imbue them with bactericidal properties. Despite this, the chemical fate of the metal in biological systems is poorly understood. Silver(I) is a soft metal with high affinity for soft donor atoms and displays much similarity to the chemistry of Cu(I). In bacteria, interaction of silver with the cell wall/membrane, DNA, and proteins and enzymes can lead to cell death. Additionally, the intracellular generation of reactive oxygen species by silver is posited to be a significant antimicrobial action. While the antibacterial action of silver is well known, bacteria found in silver mines display resistance against it through use of a protein ensemble thought to have been specifically developed for the metal, highlighting the need for judicious use. In mammals, ∼10-20% of ingested silver is retained by the body and thought to predominantly localize in the liver or kidneys. Chronic exposure can result in argyria, a condition characterized by blue staining of the skin, resulting from subdermal deposition of silver [as Ag(0)/sulfides], but more insidious side effects, such as inclusions in the brain, seizures, liver/kidney damage, and immunosuppression, have also been reported. Here, we hope to highlight the current understanding of the biological chemistry of silver and the necessity for continued study of these systems to fill existing gaps in knowledge.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

Using Cross-Correlated Spin Relaxation to Characterize Backbone Dihedral Angle Distributions of Flexible Protein Segments.

Crucial to the function of proteins is their existence as conformational ensembles sampling numerous and structurally diverse substates. Despite this widely accepted notion there is still a high demand for meaningful and reliable approaches to characterize protein ensembles in solution. As it is usually conducted in solution, NMR spectroscopy offers unique possibilities to address this challenge. Particularly, cross‐correlated relaxation (CCR) effects have long been established to encode both protein structure and dynamics in a compelling manner. However, this wealth of information often limits their use in practice as structure and dynamics might prove difficult to disentangle. Using a modern Maximum Entropy (MaxEnt) reweighting approach to interpret CCR rates of Ubiquitin, we demonstrate that these uncertainties do not necessarily impair resolving CCR‐encoded structural information. Instead, a suitable balance between complementary CCR experiments and prior information is found to be the most crucial factor in mapping backbone dihedral angle distributions. Experimental and systematic deviations such as oversimplified dynamics appear to be of minor importance. Using Ubiquitin as an example, we demonstrate that CCR rates are capable of characterizing rigid and flexible residues alike, indicating their unharnessed potential in studying disordered proteins.

Machine Learning Identifies Robust Matrisome Markers and Regulatory Mechanisms in Cancer.

The expression and regulation of matrisome genes—the ensemble of extracellular matrix, ECM, ECM-associated proteins and regulators as well as cytokines, chemokines and growth factors—is of paramount importance for many biological processes and signals within the tumor microenvironment. The availability of large and diverse multi-omics data enables mapping and understanding of the regulatory circuitry governing the tumor matrisome to an unprecedented level, though such a volume of information requires robust approaches to data analysis and integration. In this study, we show that combining Pan-Cancer expression data from The Cancer Genome Atlas (TCGA) with genomics, epigenomics and microenvironmental features from TCGA and other sources enables the identification of “landmark” matrisome genes and machine learning-based reconstruction of their regulatory networks in 74 clinical and molecular subtypes of human cancers and approx. 6700 patients. These results, enriched for prognostic genes and cross-validated markers at the protein level, unravel the role of genetic and epigenetic programs in governing the tumor matrisome and allow the prioritization of tumor-specific matrisome genes (and their regulators) for the development of novel therapeutic approaches.

Biphasic Packing of DNA and Internal Proteins in Bacteriophage T4 Heads Revealed by Bubblegram Imaging.

The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions; it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.

Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain-mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

Spatiotemporal identification of druggable binding sites using deep learning

Identification of novel protein binding sites expands druggable genome and opens new opportunities for drug discovery. Generally, presence or absence of a binding site depends on the three-dimensional conformation of a protein, making binding site identification resemble the object detection problem in computer vision. Here we introduce a computational approach for the large-scale detection of protein binding sites, that considers protein conformations as 3D-images, binding sites as objects on these images to detect, and conformational ensembles of proteins as 3D-videos to analyze. BiteNet is suitable for spatiotemporal detection of hard-to-spot allosteric binding sites, as we showed for conformation-specific binding site of the epidermal growth factor receptor, oligomer-specific binding site of the ion channel, and binding site in G protein-coupled receptor. BiteNet outperforms state-of-the-art methods both in terms of accuracy and speed, taking about 1.5 minutes to analyze 1000 conformations of a protein with ~2000 atoms.

Evaluation of CONSRANK-Like Scoring Functions for Rescoring Ensembles of Protein-Protein Docking Poses.

Scoring is a challenging step in protein–protein docking, where typically thousands of solutions are generated. In this study, we ought to investigate the contribution of consensus-rescoring, as introduced by Oliva et al. (2013) with the CONSRANK method, where the set of solutions is used to build statistics in order to identify recurrent solutions. We explore several ways to perform consensus-based rescoring on the ZDOCK decoy set for Benchmark 4. We show that the information of the interface size is critical for successful rescoring in this context, but that consensus rescoring in itself performs less well than traditional physics-based evaluation. The results of physics-based and consensus-based rescoring are partially overlapping, supporting the use of a combination of these approaches.

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

Influence of electrostatic forces on the association kinetics and conformational ensemble of an intrinsically disordered protein.

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions.

Atomistic ensembles of proteins and soft matter complexes from MD simulations and solution scattering data

Atomistic ensembles of proteins and soft matter complexes from MD simulations and solution scattering data

Structural ensemble and biological activity of DciA intrinsically disordered region

DciA is a newly discovered bacterial protein involved in loading the replicative helicase DnaB onto DNA at the initiation step of chromosome replication. Its three-dimensional structure is composed of a folded N-terminal domain (residues 1–111) resembling K Homology domains and a long disordered C-terminal tail (residues 112–157) which structure–activity relationship remains to be elucidated. In the present study on Vibrio cholerae DciA, we emphasize the importance of its disordered region to load DnaB onto DNA using surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). Then we characterize the conformational ensemble of the full-length protein using a combination of circular dichroism (CD), small angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. The atomic-level structural ensemble generated by MD simulations is in very good agreement with SAXS data. From initial conformations of the C-terminal tail without any secondary structure, our simulations bring to light several transient helical structures in this segment, which might be molecular recognition features (MoRFs) for the binding to DnaB and its recruitment and loading onto DNA.

Structures of the intrinsically disordered Aβ, tau and α-synuclein proteins in aqueous solution from computer simulations

Intrinsically disordered proteins (IDPs) play many biological roles in the human proteome ranging from vesicular transport, signal transduction to neurodegenerative diseases. The Aβ and tau proteins, and the α-synuclein protein, key players in Alzheimer’s and Parkinson’s diseases, respectively are fully disordered at the monomer level. The structural heterogeneity of the monomeric and oligomeric states and the high self-assembly propensity of these three IDPs have precluded experimental structural determination. Simulations have been used to determine the atomic structures of these IDPs. In this article, we review recent computer models to capture the equilibrium ensemble of Aβ, tau and α-synuclein proteins at different association steps in aqueous solution and present new results of the PEP-FOLD framework on α-synuclein monomer.