Protein binding hot spots and the residue-residue pairing preference: a water exclusion perspective.
ABSTRACT: BACKGROUND: A protein binding hot spot is a small cluster of residues tightly packed at the center of the interface between two interacting proteins. Though a hot spot constitutes a small fraction of the interface, it is vital to the stability of protein complexes. Recently, there are a series of hypotheses proposed to characterize binding hot spots, including the pioneering O-ring theory, the insightful 'coupling' and 'hot region' principle, and our 'double water exclusion' (DWE) hypothesis. As the perspective changes from the O-ring theory to the DWE hypothesis, we examine the physicochemical properties of the binding hot spots under the new hypothesis and compare with those under the O-ring theory. RESULTS: The requirements for a cluster of residues to form a hot spot under the DWE hypothesis can be mathematically satisfied by a biclique subgraph if a vertex is used to represent a residue, an edge to indicate a close distance between two residues, and a bipartite graph to represent a pair of interacting proteins. We term these hot spots as DWE bicliques. We identified DWE bicliques from crystal packing contacts, obligate and non-obligate interactions. Our comparative study revealed that there are abundant unique bicliques to the biological interactions, indicating specific biological binding behaviors in contrast to crystal packing. The two sub-types of biological interactions also have their own signature bicliques. In our analysis on residue compositions and residue pairing preferences in DWE bicliques, the focus was on interaction-preferred residues (ipRs) and interaction-preferred residue pairs (ipRPs). It is observed that hydrophobic residues are heavily involved in the ipRs and ipRPs of the obligate interactions; and that aromatic residues are in favor in the ipRs and ipRPs of the biological interactions, especially in those of the non-obligate interactions. In contrast, the ipRs and ipRPs in crystal packing are dominated by hydrophilic residues, and most of the anti-ipRs of crystal packing are the ipRs of the obligate or non-obligate interactions. CONCLUSIONS: These ipRs and ipRPs in our DWE bicliques describe a diverse binding features among the three types of interactions. They also highlight the specific binding behaviors of the biological interactions, sharply differing from the artifact interfaces in the crystal packing. It can be noted that DWE bicliques, especially the unique bicliques, can capture deep insights into the binding characteristics of protein interfaces.
Project description:The underlying physico-chemical principles of the interactions between domains in protein folding are similar to those between protein molecules in binding. Here we show that conserved residues and experimental hot spots at intermolecular binding interfaces overlap residues that vibrate with high frequencies. Similarly, conserved residues and hot spots are found in protein cores and are also observed to vibrate with high frequencies. In both cases, these residues contribute significantly to the stability. Hence, these observations validate the proposition that binding and folding are similar processes. In both packing plays a critical role, rationalizing the residue conservation and the experimental alanine scanning hot spots. We further show that high-frequency vibrating residues distinguish between protein binding sites and the remainder of the protein surface.
Project description:<h4>Background</h4>Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available.<h4>Results</h4>Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure.<h4>Conclusion</h4>NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at http://noxclass.bioinf.mpi-inf.mpg.de/.
Project description:CaWRKY40 was previously found to be transcriptionally up-regulated by Ralstonia solanacearum inoculation (RSI) or heat stress (HS), but the underlying mechanism remains unknown. Herein, we report that a double-W box-element (DWE) in the promoter of CaWRKY40 is critical for these responses. The upstream W box unit WI of this composite element is crucial for preferential binding by CaWRKY40 and responsiveness to RSI or HS. DWE-driven CaWRKY40 can be transcriptionally and nonspecifically regulated by itself and by CaWRKY58 and CaWRKY27. The DWE was also found in the promoters of CaWRKY40 orthologs, including AtWRKY40, VvWRKY40, GmWRKY40, CplWRKY40, SaWRKY40, SpWRKY40, NtWRKY40, and NaWRKY40. DWE<sup>AtWRKY40</sup> was analogous to DWE<sup>CaWRKY40</sup> by responding to RSI or HS and AtWRKY40 expression. These data suggest that a conserved response of plants to pathogen infection or HS is probably mediated by binding of the DWE by WRKY40.
Project description:Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a ?-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through ?-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.
Project description:Development of small molecule inhibitors of protein-protein interactions (PPIs) is hampered by our poor understanding of the druggability of PPI target sites. Here, we describe the combined application of alanine-scanning mutagenesis, fragment screening, and FTMap computational hot spot mapping to evaluate the energetics and druggability of the highly charged PPI interface between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), an important drug target. FTMap identifies four binding energy hot spots at the active site. Only two of these are exploited by Nrf2, which alanine scanning of both proteins shows to bind primarily through E79 and E82 interacting with KEAP1 residues S363, R380, R415, R483, and S508. We identify fragment hits and obtain X-ray complex structures for three fragments via crystal soaking using a new crystal form of KEAP1. Combining these results provides a comprehensive and quantitative picture of the origins of binding energy at the interface. Our findings additionally reveal non-native interactions that might be exploited in the design of uncharged synthetic ligands to occupy the same site on KEAP1 that has evolved to bind the highly charged DEETGE binding loop of Nrf2. These include π-stacking with KEAP1 Y525 and interactions at an FTMap-identified hot spot deep in the binding site. Finally, we discuss how the complementary information provided by alanine-scanning mutagenesis, fragment screening, and computational hot spot mapping can be integrated to more comprehensively evaluate PPI druggability.
Project description:The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.
Project description:Identifying features that effectively represent the energetic contribution of an individual interface residue to the interactions between proteins remains problematic. Here, we present several new features and show that they are more effective than conventional features. By combining the proposed features with conventional features, we develop a predictive model for interaction hot spots. Initially, 54 multifaceted features, composed of different levels of information including structure, sequence and molecular interaction information, are quantified. Then, to identify the best subset of features for predicting hot spots, feature selection is performed using a decision tree. Based on the selected features, a predictive model for hot spots is created using support vector machine (SVM) and tested on an independent test set. Our model shows better overall predictive accuracy than previous methods such as the alanine scanning methods Robetta and FOLDEF, and the knowledge-based method KFC. Subsequent analysis yields several findings about hot spots. As expected, hot spots have a larger relative surface area burial and are more hydrophobic than other residues. Unexpectedly, however, residue conservation displays a rather complicated tendency depending on the types of protein complexes, indicating that this feature is not good for identifying hot spots. Of the selected features, the weighted atomic packing density, relative surface area burial and weighted hydrophobicity are the top 3, with the weighted atomic packing density proving to be the most effective feature for predicting hot spots. Notably, we find that hot spots are closely related to pi-related interactions, especially pi . . . pi interactions.
Project description:Thiol peroxidases (Tpxs) are dimeric 2-Cys peroxiredoxins from bacteria that preferentially reduce alkyl hydroperoxides. Catalysis requires two conserved residues, the peroxidatic cysteine and the resolving cysteine, which are located in helix alpha(2) and helix alpha(3), respectively. The partial unraveling of helices alpha(2) and alpha(3) during catalysis allows for the formation of an intramolecular disulfide between these two residues. Here, we present three structures of Escherichia coli Tpx representing the fully folded (peroxide binding site intact), locally unfolded (disulfide bond), and partially locally unfolded (transitional state) conformations. We also compare known Tpx crystal structures and analyze the sequence-conservation patterns among nearly 300 Tpx sequences. Twelve fully conserved Tpx-specific residues cluster at the active site and dimer interface, and an additional 37 highly conserved residues are mostly located in a cradle providing the environment for helix alpha(2). Using the structures determined here as representative fully folded, transitional, and locally unfolded Tpx conformations, we describe in detail the structural changes associated with catalysis in the Tpx subfamily. Key insights include the description of a conserved hydrophobic collar around the active site, a set of conserved packing interactions between helices alpha(2) and alpha(3) that allow the local unfolding of alpha(2) to trigger the partial unfolding of alpha(3), a conserved dimer interface that anchors the ends of helices alpha(2) and alpha(3) to stabilize the active site during structural transitions, and a conserved set of residues constituting a cradle that stabilizes the two discrete conformations of helix alpha(2) involved in catalysis. The involvement of the dimer interface in stabilizing active-site folding and in forming the hydrophobic collar implies that Tpx is an obligate homodimer and explains the high conservation of interface residues.
Project description:Most biological processes involve multiple proteins interacting with each other. It has been recently discovered that certain residues in these protein-protein interactions, which are called hot spots, contribute more significantly to binding affinity than others. Hot spot residues have unique and diverse energetic properties that make them challenging yet important targets in the modulation of protein-protein complexes. Design of therapeutic agents that interact with hot spot residues has proven to be a valid methodology in disrupting unwanted protein-protein interactions. Using biological methods to determine which residues are hot spots can be costly and time consuming. Recent advances in computational approaches to predict hot spots have incorporated a myriad of features, and have shown increasing predictive successes. Here we review the state of knowledge around protein-protein interactions, hot spots, and give an overview of multiple in silico prediction techniques of hot spot residues.
Project description:Conserved residues in protein-protein interfaces correlate with residue hot-spots. To obtain insight into their roles, we have studied their mobility. We have performed 39 explicit solvent simulations of 15 complexes and their monomers, with the interfaces varying in size, shape, and function. The dynamic behavior of conserved residues in unbound monomers illustrates significantly lower flexibility as compared to their environment, suggesting that already before binding they are constrained in a boundlike configuration. To understand this behavior, we have analyzed the inter- and intrachain hydrogen-bond residence-time in the interfaces. We find that conserved residues are not involved significantly in hydrogen bonds across the interface as compared to nonconserved. However, the monomer simulations reveal that conserved residues contribute dominantly to hydrogen-bond formation before binding. Packing of conserved residues across the trajectories is significantly higher before and after the binding, rationalizing their lower mobility. Backbone torsional angle distributions show that conserved residues assume restricted regions of space and the most visited conformations in the bound and unbound trajectories are similar, suggesting that conserved residues are preorganized. Combined with previous studies, we conclude that conserved residues, hot spots, anchor, and interface-buried residues may be similar residues, fulfilling similar roles.