Changes in protein structure at the interface accompanying complex formation.

Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein-Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

A minimal model of protein-protein binding affinities.

A minimal model of protein-protein binding affinity that takes into account only two structural features of the complex, the size of its interface, and the amplitude of the conformation change between the free and bound subunits, is tested on the 144 complexes of a structure-affinity benchmark. It yields Kd values that are within two orders of magnitude of the experiment for 67% of the complexes, within three orders for 88%, and fails on 12%, which display either large conformation changes, or a very high or a low affinity. The minimal model lacks the specificity and accuracy needed to make useful affinity predictions, but it should help in assessing the added value of parameters used by more elaborate models, and set a baseline for evaluating their performances.

Structural templates for modeling homodimers.

Oligomeric proteins are more abundant in nature than monomeric proteins, and involved in all biological processes. In the absence of an experimental structure, their subunits can be modeled from their sequence like monomeric proteins, but reliable procedures to build the oligomeric assembly are scarce. Template-based methods, which start from known protein structures, are commonly applied to model subunits. We present a method to model homodimers that relies on a structural alignment of the subunits, and test it on a set of 511 target structures recently released by the Protein Data Bank, taking as templates the earlier released structures of 3108 homodimeric proteins (H-set), and 2691 monomeric proteins that form dimer-like assemblies in crystals (M-set). The structural alignment identifies a H-set template for 97% of the targets, and in half of the cases, it yields a correct model of the dimer geometry and residue-residue contacts in the target. It also identifies a M-set template for most of the targets, and some of the crystal dimers are very similar to the target homodimers. The procedure efficiently detects homology at low levels of sequence identities, and points to erroneous quaternary structures in the Protein Data Bank. The high coverage of the target set suggests that the content of the Protein Data Bank already approaches the structural diversity of protein assemblies in nature, and that template-based methods should become the choice method for modeling oligomeric as well as monomeric proteins.

The targets of CAPRI rounds 20-27.

Eight CAPRI prediction rounds with a total of 15 targets were held in the years 2010-2012. Only five of the targets were protein assemblies comparable with those of earlier CAPRI rounds. In one target, the solvent positions at the interface had to be predicted; another was a protein-polysaccharide complex. The remainders were designed complexes issued from protein engineering experiments, and the prediction concerned either their structure or the binding affinity of the designed ligand. Affinity prediction was a new experiment in CAPRI, and a challenge for its participants. It pushed the community into developing novel procedures and score functions that will improve the performance of docking methods, help designing binders, and yield better structure-based estimates of the binding free energy of natural assemblies.

Reassessing buried surface areas in protein-protein complexes.

The buried surface area (BSA), which measures the size of the interface in a protein-protein complex may differ from the accessible surface area (ASA) lost upon association (which we call DSA), if conformation changes take place. To evaluate the DSA, we measure the ASA of the interface atoms in the bound and unbound states of the components of 144 protein-protein complexes taken from the Protein-Protein Interaction Affinity Database of Kastritis et al. (2011). We observe differences exceeding 20%, and a systematic bias in the distribution. On average, the ASA calculated in the bound state of the components is 3.3% greater than in their unbound state, and the BSA, 7% greater than the DSA. The bias is observed even in complexes where the conformation changes are small. An examination of the bound and unbound structures points to a possible origin: local movements optimize contacts with the other component at the cost of internal contacts, and presumably also the binding free energy.

Protein flexibility, not disorder, is intrinsic to molecular recognition.

An 'intrinsically disordered protein' (IDP) is assumed to be unfolded in the cell and perform its biological function in that state. We contend that most intrinsically disordered proteins are in fact proteins waiting for a partner (PWPs), parts of a multi-component complex that do not fold correctly in the absence of other components. Flexibility, not disorder, is an intrinsic property of proteins, exemplified by X-ray structures of many enzymes and protein-protein complexes. Disorder is often observed with purified proteins in vitro and sometimes also in crystals, where it is difficult to distinguish from flexibility. In the crowded environment of the cell, disorder is not compatible with the known mechanisms of protein-protein recognition, and, foremost, with its specificity. The self-assembly of multi-component complexes may, nevertheless, involve the specific recognition of nascent polypeptide chains that are incompletely folded, but then disorder is transient, and it must remain under the control of molecular chaperones and of the quality control apparatus that obviates the toxic effects it can have on the cell.

Templates are available to model nearly all complexes of structurally characterized proteins.

Traditional approaches to protein-protein docking sample the binding modes with no regard to similar experimentally determined structures (templates) of protein-protein complexes. Emerging template-based docking approaches utilize such similar complexes to determine the docking predictions. The docking problem assumes the knowledge of the participating proteins' structures. Thus, it provides the possibility of aligning the structures of the proteins and the template complexes. The progress in the development of template-based docking and the vast experience in template-based modeling of individual proteins show that, generally, such approaches are more reliable than the free modeling. The key aspect of this modeling paradigm is the availability of the templates. The current common perception is that due to the difficulties in experimental structure determination of protein-protein complexes, the pool of docking templates is insignificant, and thus a broad application of template-based docking is possible only at some future time. The results of our large scale, systematic study show that, surprisingly, in spite of the limited number of protein-protein complexes in the Protein Data Bank, docking templates can be found for complexes representing almost all the known protein-protein interactions, provided the components themselves have a known structure or can be homology-built. About one-third of the templates are of good quality when they are compared to experimental structures in test sets extracted from the Protein Data Bank and would be useful starting points in modeling the complexes. This finding dramatically expands our ability to model protein interactions, and has far-reaching implications for the protein docking field in general.

A survey of hemoglobin quaternary structures.

We perform an analysis of the quaternary structure and dimer/dimer interface in the crystal structures of 165 human hemoglobin tetramers; 112 are in the T, 17 the R, 14 the Y (or R2) state; 11 are high-affinity T state mutants, and 11 may either be intermediates between the states, or off the allosteric transition pathway. The tertiary structure is fixed within each state, in spite of the different ligands, mutations, and chemical modifications present in individual entries. The geometry of the tetramer assembly is essentially the same in all the R or the Y state entries; it is slightly different in high salt and low salt crystals of T state hemoglobins. The dimer/dimer interface differs in terms of size, chemical composition and polar interactions, between the states. It is loosely packed, like crystal packing contacts or the subunit interface of weakly associated homodimers, and unlike most oligomeric proteins, which have close-packed interfaces. The loose packing is most obvious in the liganded forms, where the tetramer is known to dissociate at low concentration. We identify cavities that contribute to the loose packing of the α1ß2 and α2ß1 contacts. Two pairs of cavities occur recurrently in both the T and the R state tetramers. They may contribute to the allosteric mechanism by facilitating the subunit movements and the tertiary structure changes that accompany the transition from T to R to Y.

A structure-based benchmark for protein-protein binding affinity.

We have assembled a nonredundant set of 144 protein-protein complexes that have high-resolution structures available for both the complexes and their unbound components, and for which dissociation constants have been measured by biophysical methods. The set is diverse in terms of the biological functions it represents, with complexes that involve G-proteins and receptor extracellular domains, as well as antigen/antibody, enzyme/inhibitor, and enzyme/substrate complexes. It is also diverse in terms of the partners' affinity for each other, with K(d) ranging between 10(-5) and 10(-14) M. Nine pairs of entries represent closely related complexes that have a similar structure, but a very different affinity, each pair comprising a cognate and a noncognate assembly. The unbound structures of the component proteins being available, conformation changes can be assessed. They are significant in most of the complexes, and large movements or disorder-to-order transitions are frequently observed. The set may be used to benchmark biophysical models aiming to relate affinity to structure in protein-protein interactions, taking into account the reactants and the conformation changes that accompany the association reaction, instead of just the final product.

Protein-protein docking benchmark version 4.0.

We updated our protein-protein docking benchmark to include complexes that became available since our previous release. As before, we only considered high-resolution complex structures that are nonredundant at the family-family pair level, for which the X-ray or NMR unbound structures of the constituent proteins are also available. Benchmark 4.0 adds 52 new complexes to the 124 cases of Benchmark 3.0, representing an increase of 42%. Thus, benchmark 4.0 provides 176 unbound-unbound cases that can be used for protein-protein docking method development and assessment. Seventeen of the newly added cases are enzyme-inhibitor complexes, and we found no new antigen-antibody complexes. Classifying the new cases according to expected difficulty for protein-protein docking algorithms gives 33 rigid body cases, 11 cases of medium difficulty, and 8 cases that are difficult. Benchmark 4.0 listings and processed structure files are publicly accessible at http://zlab.umassmed.edu/benchmark/.

Side-chain rotamer transitions at protein-protein interfaces.

We compare the changes in side chain conformations that accompany the formation of protein-protein complexes, in residues forming either the interface or the remainder of the solvent-accessible surface of the proteins in the Docking Benchmark 3.0. We find that the interface residues undergo significantly more changes than other surface residues, and these changes are more likely to convert them from a high-energy torsion angle state to a lower-energy one than the reverse. Moreover, in both the unbound proteins and the complexes, the interface residues are more frequently found to be in a high-energy torsion angle state than the noninterface residues. As these differences exist before the binding step, they may be relevant to specificity and help in identifying binding sites for docking predictions.

Protein-protein docking tested in blind predictions: the CAPRI experiment.

Docking algorithms build multimolecular assemblies based on the subunit structures. "Unbound" docking, which starts with the free molecules and allows for conformation changes, may be used to predict the structure of a protein-protein complex. This requires at least two steps, a rigid-body search that determines the relative position and orientation of the subunits, and a refinement step. The methods developed in the past twenty years yield native-like models in most cases, but always with many false positives that must be filtered out, and they fail when the conformation changes are large. CAPRI (Critical Assessment of PRedicted Interactions) is a community-wide experiment set up to monitor progress in the field. It offers participants the opportunity to test their methods in blind predictions that are assessed against an unpublished experimental structure. The models submitted by predictor groups are judged depending on how well they reproduce the geometry and the residue-residue contacts seen in the target structure. In nine years of CAPRI, 42 target complexes have been subjected to prediction based on the components' unbound structures. Good models have been submitted for 28 targets, and prediction has failed on 6. Both these successes and these failures have been fruitful, as they stimulated participant groups to develop new score functions to identify native-like solutions, and new algorithms that allow the molecules to be flexible during docking.

The targets of CAPRI Rounds 13-19.

Seven rounds of CAPRI predictions with a total of 14 targets were held in the period June 2007-November 2009. In addition to protease/inhibitor complexes and complexes with G-proteins, some of the targets displayed novel features presenting new challenges to the predictors: a complex with RNA, a leucine zipper to be built ab initio, and a human-designed protein. Nine targets were unbound or required model building; the other five had a bound component. Thirteen were assessed, and the results show that the predictor and scorer groups submitted two- or three-star (medium or high quality) models for eight of them, one-star (acceptable) models for three but failed on the unbound RNA complex and another unbound target.

Human and viral nucleoside/nucleotide kinases involved in antiviral drug activation: structural and catalytic properties.

Antiviral nucleoside and nucleotide analogs, essential for the treatment of viral infections in the absence of efficient vaccines, are prodrug forms of the active compounds that target the viral DNA polymerase or reverse transcriptase. The activation process requires several successive phosphorylation steps catalyzed by different kinases, which are present in the host cell or encoded by some of the viruses. These activation reactions often are rate-limiting steps and are thus open to improvement. We review here the structural and enzymatic properties of the enzymes that carry out the activation of analogs used in therapy against human immunodeficiency virus and against DNA viruses such as hepatitis B, herpes and poxviruses. Four major classes of drugs are considered: thymidine analogs, non-natural L-nucleosides, acyclic nucleoside analogs and acyclic nucleoside phosphonate analogs. Their efficiency as drugs depends both on the low specificity of the viral polymerase that allows their incorporation into DNA, but also on the ability of human/viral kinases to provide the activated triphosphate active forms at a high concentration at the right place. Two distinct modes of action are considered, depending on the origin of the kinase (human or viral). If the human kinases are house-keeping enzymes that belong to the metabolic salvage pathway, herpes and poxviruses encode for related enzymes. The structures, substrate specificities and catalytic properties of each of these kinases are discussed in relation to drug activation.

Analysis and prediction of protein quaternary structure.

The quaternary structure (QS) of a protein is determined by measuring its molecular weight in solution. The data have to be extracted from the literature, and they may be missing even for proteins that have a crystal structure reported in the Protein Data Bank (PDB). The PDB and other databases derived from it report QS information that either was obtained from the depositors or is based on an analysis of the contacts between polypeptide chains in the crystal, and this frequently differs from the QS determined in solution.The QS of a protein can be predicted from its sequence using either homology or threading methods. However, a majority of the proteins with less than 30% sequence identity have different QSs. A model of the QS can also be derived by docking the subunits when their 3D structure is independently known, but the model is likely to be incorrect if large conformation changes take place when the oligomer assembles.

The subunit interfaces of weakly associated homodimeric proteins.

We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

Nucleoside diphosphate kinase and the activation of antiviral phosphonate analogs of nucleotides: binding mode and phosphorylation of tenofovir derivatives.

Tenofovir is an acyclic phosphonate analog of deoxyadenylate used in AIDS and hepatitis B therapy. We find that tenofovir diphosphate, its active form, can be produced by human nucleoside diphosphate kinase (NDPK), but with low efficiency, and that creatine kinase is significantly more active. The 1.65 A x-ray structure of NDPK in complex with tenofovir mono- and diphosphate shows that the analogs bind at the same site as natural nucleotides, but in a different conformation, and make only a subset of the Van der Waals and polar interactions made by natural substrates, consistent with their comparatively low affinity for the enzyme.

Protein-protein interaction and quaternary structure.

Protein-protein recognition plays an essential role in structure and function. Specific non-covalent interactions stabilize the structure of macromolecular assemblies, exemplified in this review by oligomeric proteins and the capsids of icosahedral viruses. They also allow proteins to form complexes that have a very wide range of stability and lifetimes and are involved in all cellular processes. We present some of the structure-based computational methods that have been developed to characterize the quaternary structure of oligomeric proteins and other molecular assemblies and analyze the properties of the interfaces between the subunits. We compare the size, the chemical and amino acid compositions and the atomic packing of the subunit interfaces of protein-protein complexes, oligomeric proteins, viral capsids and protein-nucleic acid complexes. These biologically significant interfaces are generally close-packed, whereas the non-specific interfaces between molecules in protein crystals are loosely packed, an observation that gives a structural basis to specific recognition. A distinction is made within each interface between a core that contains buried atoms and a solvent accessible rim. The core and the rim differ in their amino acid composition and their conservation in evolution, and the distinction helps correlating the structural data with the results of site-directed mutagenesis and in vitro studies of self-assembly.