Folding and binding cascades: dynamic landscapes and population shifts

Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713-720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181-1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970-9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three-dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings.     

Evaluating conformational changes in protein structures binding RNA

Many protein-RNA recognition events are known to exhibit conformational changes from qualitative observations of individual complexes. However, a quantitative estimation of conformational changes is required if protein-RNA docking and template-based methods for RNA binding site prediction are to be developed. This study presents the first quantitative evaluation of conformational changes that occur when proteins bind RNA. The analysis of twelve RNA-binding proteins in the bound and unbound states using error-scaled difference distance matrices is presented. The binding site residues are mapped to each structure, and the conformational changes that affect these residues are evaluated. Of the twelve proteins four exhibit greater movements in nonbinding site residues, and a further four show the greatest movements in binding site residues. The remaining four proteins display no significant conformational change. When interface residues are found to be in conformationally variable regions of the protein they are typically seen to move less than 2 A between the bound and unbound conformations. The current data indicate that conformational changes in the binding site residues of RNA binding proteins may not be as significant as previously suggested, but a larger data set is required before wider conclusions may be drawn. The implications of the observed conformational changes for protein function prediction are discussed.     

Pre-docking filter for protein and ligand 3D structures

Virtual drug screening using protein-ligand docking techniques is a time-consuming process, which requires high computational power for binding affinity calculation. There are millions of chemical compounds available for docking. Eliminating compounds that are unlikely to exhibit high binding affinity from the screening set should speed-up the virtual drug screening procedure. We performed docking of 6353 ligands against twenty-one protein X-ray crystal structures. The docked ligands were ranked according to their calculated binding affinities, from which the top five hundred and the bottom five hundred were selected. We found that the volume and number of rotatable bonds of the top five hundred docked ligands are similar to those found in the crystal structures and corresponded with the volume of the binding sites. In contrast, the bottom five hundred set contains ligands that are either too large to enter the binding site, or too small to bind with high specificity and affinity to the binding site. A pre-docking filter that takes into account shapes and volumes of the binding sites as well as ligand volumes and flexibilities can filter out low binding affinity ligands from the screening sets. Thus, the virtual drug screening procedure speed is increased.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

Conformational diversity of ligands bound to proteins

The phenomenon of molecular recognition, which underpins almost all biological processes, is dynamic, complex and subtle. Establishing an interaction between a pair of molecules involves mutual structural rearrangements guided by a highly convoluted energy landscape, the accurate mapping of which continues to elude us. Increased understanding of the degree to which the conformational space of a ligand is restricted upon binding may have important implications for docking studies, structure refinement and for function prediction methods based on geometrical comparisons of ligands or their binding sites. Here, we present an analysis of the conformational variability exhibited by three of the most ubiquitous biological ligands in nature, ATP, NAD and FAD. First, we demonstrate qualitatively that these ligands bind to proteins in widely varying conformations, including several cases in which parts of the molecule assume energetically unfavourable orientations. Next, by comparing the distribution of bound ligand shapes with the set of all possible molecular conformations, we provide a quantitative assessment of previous observations that ligands tend to unfold when binding to proteins. We show that, while extended forms of ligands are indeed common in ligand-protein structures, instances of ligands in almost maximally compact arrangements can also be found. Thirdly, we compare the conformational variation in two sets of ligand molecules, those bound to homologous proteins, and those bound to unrelated proteins. Although most superfamilies bind ligands in a fairly conserved manner, we find several cases in which significant variation in ligand configuration is observed.     

Heme proteins—Diversity in structural characteristics,function, and folding

The characteristics of heme prosthetic groups and their binding sites have been analyzed in detail in a data set of nonhomologous heme proteins. Variations in the shape, volume, and chemical composition of the binding site, in the mode of heme binding and in the number and nature of heme–protein interactions are found to result in significantly different heme environments in proteins with different functions in biology. Differences are also seen in the properties of the apo states of the proteins. The apo states of proteins that bind heme permanently in their functional form show some disorder, ranging from local unfolding in the heme binding pocket to complete unfolding to give a random coil. In contrast, proteins that bind heme transiently are fully folded in their apo and holo states, presumably allowing both apo and holo forms to remain biologically active resisting aggregation or proteolysis. The principles identified here provide a framework for the design of de novo proteins that will exhibit tight heme ligand binding and for the identification of the function of structural genomic target proteins with heme ligands. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Ligand binding to a high‐energy partially unfolded protein

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP⁺ as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP⁺ and found that NADP⁺ binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP⁺ is diminished upon partial unfolding. Based on known crystallographic structures of NADP⁺‐bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine‐binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP⁺. Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high‐energy non‐native forms.     

Cofactor‐binding sites in proteins of deviating sequence: Comparative analysis and clustering in torsion angle,cavity, and fold space

Small molecules are recognized in protein‐binding pockets through surface‐exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein–ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise tounexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein–cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein‐binding pockets, and the local folding patterns next to the cofactor‐binding site. State‐of‐the‐art clustering techniques have been applied to group the different protein–cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Structural analysis of protein tyrosine phosphatase 1B reveals potentially druggable allosteric binding sites

Catalytic proteins such as human protein tyrosine phosphatase 1B (PTP1B), with conserved and highly polar active sites, warrant the discovery of druggable nonactive sites, such as allosteric sites, and potentially, therapeutic small molecules that can bind to these sites. Catalyzing the dephosphorylation of numerous substrates, PTP1B is physiologically important in intracellular signal transduction pathways in diverse cell types and tissues. Aberrant PTP1B is associated with obesity, diabetes, cancers, and neurodegenerative disorders. Utilizing clustering methods (based on root mean square deviation, principal component analysis, nonnegative matrix factorization, and independent component analysis), we have examined multiple PTP1B structures. Using the resulting representative structures in different conformational states, we determined consensus clustroids and used them to identify both known and novel binding sites, some of which are potentially allosteric. We report several lead compounds that could potentially bind to the novel PTP1B binding sites and can be further optimized. Considering the possibility for drug repurposing, we discovered homologous binding sites in other proteins, with ligands that could potentially bind to the novel PTP1B binding sites.     

Siderocalin Q83 exhibits differential slow dynamics upon ligand binding

Siderocalin Q83 is a small soluble protein that has the ability to bind two different ligands (enterobactin and arachidonic acid) simultaneously in two distinct binding sites. Here we report that Q83 exhibits an intriguing dynamic behavior. In its free form, the protein undergoes significant micro-to-millisecond dynamics. When binding arachidonic acid, the motions of the arachidonic acid binding site are quenched while the dynamics at the enterobactin binding site increases. Reciprocally, enterobactin binding to Q83 quenches the motions at the enterobactin binding site and increases the slow dynamics at the arachidonic acid binding site. Additionally, in the enterobactin-bound state, the excited state of the arachidonic acid binding site resembles the arachidonic acid-bound state. These observations strongly suggest an allosteric regulation where binding of one ligand enhances the affinity of Q83 for the other one. Additionally, our data strengthen the emerging view of proteins as dynamic ensembles interconverting between different sub-states with distinct functionalities.     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.     

Classification of proteins based on the properties of the ligand-binding site: the case of adenine-binding proteins

Comparative analysis of protein binding sites for similar ligands yields information about conserved interactions, relevant for ligand affinity, and variable interactions, which are important for specificity. The pattern of variability can indicate new targets for a pharmacologically validated class of compounds binding to a similar site. A particularly vast group of therapeutically interesting proteins using the same or similar substrates are those that bind adenine-containing ligands. Drug development is focusing on compounds occupying the adenine-binding site and their specificity is an issue of paramount importance. We use a simple scheme to characterize and classify the adenine-binding sites in terms of their intermolecular interactions, and show that this classification does not necessarily correspond to protein classifications based on either sequence or structural similarity. We find that only a limited number of the different hydrogen bond patterns possible for adenine-binding is used, which can be utilized as an effective classification scheme. Closely related protein families usually share similar hydrogen patterns, whereas non-polar interactions are less well conserved. Our classification scheme can be used to select groups of proteins with a similar ligand-binding site, thus facilitating the definition of the properties that can be exploited to design specific inhibitors.     

The aromatic circular dichroism spectrum as a probe for conformational changes in the active site environment of hemocyanins.

The binding of various ligand molecules to the binuclear Cu(I) site of deoxy-hemocyanin has been investigated through the changes produced in the aromatic region of the circular dichroism spectrum of the protein, where a cluster of tryptophan residues located in the vicinity of copper site undergo conformational reorientations in the presence of exogenous ligands coordinated to the metal. In agreement with expectations, the binuclear site of arthropod hemocyanin is severely hindered to the access of exogenous ligands except for very small molecules like CO, O2 or CN- while for mollusc proteins ligands such as thiourea and 2-mercaptoethanol bind easily to the Cu(I) sites. However, the access of the ligand becomes progressively hindered and eventually prevented as the size of substituents on the ligand increases.     

Analysis of the structure of human apo-S100B at low temperature indicates a unimodal conformational distribution is adopted by calcium-free S100 proteins

S100B is one of the best-characterized members of the calcium-signaling S100 protein family. Most S100 proteins are dimeric, with each monomer containing two EF-hand calcium-binding sites (EF1, EF2). S100B and other S100 proteins respond to calcium increases in the cell by coordinating calcium and undergoing a conformational change that allows them to interact with a variety of cellular targets. Although several three dimensional structures of S100 proteins are available in the calcium-free (apo-) state it has been observed that these structures appear to adopt a wide range of conformations in the EF2 site with respect to the positioning of helix III, the helix that undergoes the most dramatic calcium-induced conformational change. In this work, we have determined the structure of human apo-S100B at 10 degrees C to examine whether temperature might be responsible for these structural differences. Further, we have used this data, and other available apo-S100 structures, to show that despite the range of interhelical angles adopted in the apo-S100 structures, normal Gaussian distributions about the mean angles found in the structure of human apo-S100B are observed. This finding, only obvious from the analysis of all available apo-S100 proteins, provides direct structural evidence that helix III is a loosely packed helix. This is likely a necessary functional property of the S100 proteins that facilitates the calcium-induced conformational change of helix III. In contrast, the calcium-bound structures of the S100 proteins show significantly smaller variability in the interhelical angles. This shows that calcium binding to the S100 proteins causes not only a conformational change but results in a tighter distribution of helices within the EF2 calcium binding site required for target protein interactions.     

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl‐tRNA synthetase enzymes

Orthogonal aminoacyl‐tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM‐based molecular docking and free energy calculations were used to evaluate rational design of specific protein–ligand interactions for aminoacyl‐tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p‐benzoyl‐L ‐phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man‐α‐O‐Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interactions in native binding sites cause a large change in protein dynamics

Cellular functions are regulated by molecules that interact with proteins and alter their activities. To enable such control, protein activity, and therefore protein conformational distributions, must be susceptible to alteration by molecular interactions at functional sites. Here we investigate whether interactions at functional sites cause a large change in the protein conformational distribution. We apply a computational method, called dynamics perturbation analysis (DPA), to identify sites at which interactions have a large allosteric potential D(x), which is the Kullback-Leibler divergence between protein conformational distributions with and without an interaction. In DPA, a protein is decorated with surface points that interact with neighboring protein atoms, and D(x) is calculated for each of the points in a coarse-grained model of protein vibrations. We use DPA to examine hundreds of protein structures from a standard small-molecule docking test set, and find that ligand-binding sites have elevated values of D(x): for 95% of proteins, the probability of randomly obtaining values as high as those in the binding site is 10(-3) or smaller. We then use DPA to develop a computational method to predict functional sites in proteins, and find that the method accurately predicts ligand-binding-site residues for proteins in the test set. The performance of this method compares favorably with that of a cleft analysis method. The results confirm that interactions at small-molecule binding sites cause a large change in the protein conformational distribution, and motivate using DPA for large-scale prediction of functional sites in proteins. They also suggest that natural selection favors proteins whose activities are capable of being regulated by molecular interactions.     

Shape variation in protein binding pockets and their ligands

A common assumption about the shape of protein binding pockets is that they are related to the shape of the small ligand molecules that can bind there. But to what extent is that assumption true? Here we use a recently developed shape matching method to compare the shapes of protein binding pockets to the shapes of their ligands. We find that pockets binding the same ligand show greater variation in their shapes than can be accounted for by the conformational variability of the ligand. This suggests that geometrical complementarity in general is not sufficient to drive molecular recognition. Nevertheless, we show when considering only shape and size that a significant proportion of the recognition power of a binding pocket for its ligand resides in its shape. Additionally, we observe a "buffer zone" or a region of free space between the ligand and protein, which results in binding pockets being on average three times larger than the ligand that they bind.