Understanding mechanisms governing protein-protein interactions from synthetic binding interfaces

Recent advances in methodologies and design of combinatorial library selection have enabled comprehensive characterization of sequence space for protein-protein interaction interfaces and generation of fully synthetic binding interfaces. By exhaustively introducing and quantitatively analyzing mutations in natural interfaces, new insights into their molecular architecture and plasticity have emerged. Minimalist combinatorial libraries based on a restricted amino acid code have produced synthetic interfaces that rival natural ones using a different set of rules. A two amino acid code composed of just tyrosine and serine in the context of antibody CDR loops is sufficient to produce high affinity and specific interactions with different classes of protein targets. Structural analyses highlight the dominant role of Tyr in forming productive interactions and demonstrate the dominance of conformational diversity over chemical diversity in producing na?ve binding interfaces. Synthetic binding proteins are beginning to be used as a powerful crystallization tool to attack important structural biology problems that are recalcitrant to crystallization using traditional methods.     

Molecular principles of the interactions of disordered proteins

Thorough knowledge of the molecular principles of protein-protein recognition is essential to our understanding of protein function at the cellular level. Whereas interactions of ordered proteins have been analyzed in great detail, complexes of intrinsically unstructured/disordered proteins (IUPs) have hardly been addressed so far. Here, we have collected a database of 39 complexes of experimentally verified IUPs, and compared their interfaces with those of 72 complexes of ordered, globular proteins. The characteristic differences found between the two types of complexes suggest that IUPs represent a distinct molecular implementation of the principles of protein-protein recognition. The interfaces do not differ in size, but those of IUPs cover a much larger part of the surface of the protein than for their ordered counterparts. Moreover, IUP interfaces are significantly more hydrophobic relative to their overall amino acid composition, but also in absolute terms. They rely more on hydrophobic-hydrophobic than on polar-polar interactions. Their amino acids in the interface realize more intermolecular contacts, which suggests a better fit with the partner due to induced folding upon binding that results in a better adaptation to the partner. The two modes of interaction also differ in that IUPs usually use only a single continuous segment for partner binding, whereas the binding sites of ordered proteins are more segmented. Probably, all these features contribute to the increased evolutionary conservation of IUP interface residues. These noted molecular differences are also manifested in the interaction energies of IUPs. Our approximation of these by low-resolution force-fields shows that IUPs gain much more stabilization energy from intermolecular contacts, than from folding, i.e. they use their binding energy for folding. Overall, our findings provide a structural rationale to the prior suggestions that many IUPs are specialized for functions realized by protein-protein interactions.     

功能蛋白微阵列的作用

对蛋白质阵列的两大功能—分子识别和酶活测定进行了阐述.其中前者包括蛋白质-D N A间的相互作用、蛋白质-蛋白质复合体的相互作用、蛋白质-小分子之间的相互作用;后者包括阵列蛋白作为底物来测酶活、阵列蛋白作为酶的酶活.     

Ankyrin repeat: a unique motif mediating protein-protein interactions

Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.     

Functional integrative levels in the human interactome recapitulate organ organization

Interactome networks represent sets of possible physical interactions between proteins. They lack spatio-temporal information by construction. However, the specialized functions of the differentiated cell types which are assembled into tissues or organs depend on the combinatorial arrangements of proteins and their physical interactions. Is tissue-specificity, therefore, encoded within the interactome? In order to address this question, we combined protein-protein interactions, expression data, functional annotations and interactome topology. We first identified a subnetwork formed exclusively of proteins whose interactions were observed in all tested tissues. These are mainly involved in housekeeping functions and are located at the topological center of the interactome. This ‘Largest Common Interactome Network’ represents a ‘functional interactome core’. Interestingly, two types of tissue-specific interactions are distinguished when considering function and network topology: tissue-specific interactions involved in regulatory and developmental functions are central whereas tissue-specific interactions involved in organ physiological functions are peripheral. Overall, the functional organization of the human interactome reflects several integrative levels of functions with housekeeping and regulatory tissue-specific functions at the center and physiological tissue-specific functions at the periphery. This gradient of functions recapitulates the organization of organs, from cells to organs. Given that several gradients have already been identified across interactomes, we propose that gradients may represent a general principle of protein-protein interaction network organization.     

High-affinity binders selected from designed ankyrin repeat protein libraries

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.     

On the lack of specificity of proteins and its consequences for a theory of biological organization

It is now widely recognized that gene expression and cellular processes include a probabilistic component. However, this does not essentially modify the theory of genetic programming. This stochastic aspect, which is called noise, is usually conceived as a margin of fluctuation in the way the genetic program functions and the latter remains understood as a specific mechanism guided by genetic information. In contrast, recent data show that proteins do not possess a high level of specificity. They can interact with numerous molecular partners. As a consequence molecular interactions are not simply “noisy”. Because they are subject to large combinatorial interaction possibilities, they are also intrinsically stochastic and must be sorted out by the cell structure. This contradicts the genetic programming theory which is based on the idea that protein interactions are directed by their stereospecificity and genetic information. Taking into account the lack of protein specificity leads to a new theory. Natural selection acts not only in evolution but also in ontogenesis by sorting stochastic molecular interactions. In this frame, the making up of an organism, instead of being a simple bottom-top process in which information flows from genes to phenotypes, is both a bottom-top and top-bottom process. Genes provide proteins, but their stochastic interactions are sorted by selective constraints arising from the cell and multi-cellular structures, which are themselves subject to the action of natural selection.     

AutoMatch: target-binding protein design and enzyme design by automatic pinpointing potential active sites in available protein scaffolds

Proteins perform their functions mainly via active sites, whereas other parts of the proteins comprise the scaffolds, which support the active sites. One strategy for protein functional design is transplanting active sites, such as catalytic sites for enzyme or binding hot spots for protein-protein interactions, onto a new scaffold. AutoMatch is a new program designed for efficiently elucidating suitable scaffolds and potential sites on the scaffolds. Backrub motions are used to treat backbone flexibility during the design process. A step-by-step checking strategy and cluster-representation examination strategy were developed to solve the large combinatorial problem for the matching of active-site conformations. In addition, a grid-based binding energy scoring method was used to filter the solutions. An enzyme design benchmark and a protein-protein interaction design benchmark were built to test the algorithm. AutoMatch could identify the hot spots in the nonbinding protein and rank them within the top five results for 8 of 10 target-binding protein design cases. In addition, among the 15 enzymes tested, AutoMatch can identify the catalytic active sites in the apoprotein and rank them within the top five results for 13 cases. AutoMatch was also tested for screening scaffold library in designing binding proteins targeting influenza hemagglutinin, HIV gp120, and epidermal growth factor receptor kinase, respectively. AutoMatch, and the two test sets, ActApo and ActFree, are available for noncommercial applications at http://mdl.ipc.pku.edu.cn/cgi-bin/down.cgi.     

Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.     

Convergent evolution with combinatorial peptides

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.     

Advances in the study of SR protein family

The name of SR proteins is derived from their typical RS domain that is rich in serine (Ser, S) and arginine (Arg, R). They are conserved in evolution. Up to now, 10 members of the SR protein family have been identified in humans. SR proteins contain one or two RNA binding motifs aside from the RS domain, and also possess special biochemical and immunological features. As to the functions of SR proteins, they facilitate the recruitment of the components of splicesome via protein-protein interaction to prompt the assembly of early splicesome; while in alternative splicing, tissue-specifically expressed SR protein along with the relative ratio of SR protein and heterogeneous nuclear ribonucleoprotein (hnRNP) is composed of two main regulative mechanisms for alternative splicing. Almost all of the biochemical functions are regulated by reversible phosphorylation.     

Phage display: a useful tool for malaria research?

Defining the molecular intricacies of malaria pathogenesis is a vital area of medical and scientific research. Sophisticated methods have been developed to identify and characterise host-parasite interactions that are important in infection. Phage display involves the combinatorial display of proteins or peptides on the surface of bacteriophage. The technology provides an invaluable tool for screening diverse libraries for polypeptides that have a high affinity for a given target. Phage display in malaria research has proven successful, not only in mapping the protein-protein interactions that are important in Plasmodium biology, but also in the identification of molecules that might be exploited in the design of therapeutic agents or vaccines.     

Specificity of molecular interactions in transient protein-protein interaction interfaces

In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of antibody-antigen complexes, the sign is somewhat ambiguous. From the evolutionary perspective, while protease-inhibitors and sig-naling proteins have optimized their interfaces to suit their biological functions, antibody-antigen interactions are the happenstance, implying that antibody-antigen complexes do not show distinctive interaction types. Persistent interaction types such as pi...pi, amide-carbonyl, and hydroxyl-carbonyl interaction, are also investigated. Analyzing the structural orientations of the pi...pi stacking interactions, we find that herringbone shape is a major configuration in transient protein-protein interfaces. This result is different from that of protein core, where parallel-displaced configurations are the major configuration. We also analyze overall trend of amide-carbonyl and hydroxyl-carbonyl interactions. It is noticeable that nearly 82% of the interfaces have at least one hydroxyl-carbonyl interactions.     

Hot regions in protein--protein interactions: the organization and contribution of structurally conserved hot spot residues

Structurally conserved residues at protein-protein interfaces correlate with the experimental alanine-scanning hot spots. Here, we investigate the organization of these conserved, computational hot spots and their contribution to the stability of protein associations. We find that computational hot spots are not homogeneously distributed along the protein interfaces; rather they are clustered within locally tightly packed regions. Within the dense clusters, they form a network of interactions and consequently their contributions to the stability of the complex are cooperative; however the contributions of independent clusters are additive. This suggests that the binding free energy is not a simple summation of the single hot spot residue contributions. As expected, around the hot spot residues we observe moderately conserved residues, further highlighting the crucial role of the conserved interactions in the local densely packed environment. The conserved occurrence of these organizations suggests that they are advantageous for protein-protein associations. Interestingly, the total number of hydrogen bonds and salt bridges contributed by hot spots is as expected. Thus, H-bond forming residues may use a "hot spot for water exclusion" mechanism. Since conserved residues are located within highly packed regions, water molecules are easily removed upon binding, strengthening electrostatic contributions of charge-charge interactions. Hence, the picture that emerges is that protein-protein associations are optimized locally, with the clustered, networked, highly packed structurally conserved residues contributing dominantly and cooperatively to the stability of the complex. When addressing the crucial question of "what are the preferred ways of proteins to associate", these findings point toward a critical involvement of hot regions in protein-protein interactions.     

Regulatory role of SH3 domain-mediated protein-protein interactions in synaptic vesicle endocytosis.

Src homology (SH) 3 domains are small modules found in a diverse array of proteins. The presence of an SH3 domain confers upon its resident protein the ability to interact with specific proline-rich sequences in protein binding partners. A major focus of research has highlighted a role for SH3 domain-mediated interactions in the regulation of signal transduction events. However, more recent data has suggested an important function for SH3 domains in vesicular trafficking. This review will focus on this newly emerging role with a particular emphasis on the molecular components involved in synaptic vesicle endocytosis and the regulatory role of SH3 domain-mediated protein-protein interactions in this process.     

First-step mutations for adaptation at elevated temperature increase capsid stability in a virus

The relationship between mutation, protein stability and protein function plays a central role in molecular evolution. Mutations tend to be destabilizing, including those that would confer novel functions such as host-switching or antibiotic resistance. Elevated temperature may play an important role in preadapting a protein for such novel functions by selecting for stabilizing mutations. In this study, we test the stability change conferred by single mutations that arise in a G4-like bacteriophage adapting to elevated temperature. The vast majority of these mutations map to interfaces between viral coat proteins, suggesting they affect protein-protein interactions. We assess their effects by estimating thermodynamic stability using molecular dynamic simulations and measuring kinetic stability using experimental decay assays. The results indicate that most, though not all, of the observed mutations are stabilizing.     

DIP: the database of interacting proteins

The Database of Interacting Proteins (DIP; http://dip.doe-mbi.ucla.edu) is a database that documents experimentally determined protein-protein interactions. This database is intended to provide the scientific community with a comprehensive and integrated tool for browsing and efficiently extracting information about protein interactions and interaction networks in biological processes. Beyond cataloging details of protein-protein interactions, the DIP is useful for understanding protein function and protein-protein relationships, studying the properties of networks of interacting proteins, benchmarking predictions of protein-protein interactions, and studying the evolution of protein-protein interactions.     

Strategies to search and design stabilizers of protein-protein interactions: a feasibility study

Since protein-protein interactions play a pivotal role in the communication on the molecular level in virtually every biological system and process, the search and design for modulators of such interactions is of utmost importance. In recent years many inhibitors for specific protein-protein interactions have been developed, however, in only a few cases, small and druglike molecules are able to interfere in the complex formation of proteins. On the other hand, there are several small molecules known to modulate protein-protein interactions by means of stabilizing an already assembled complex. To achieve this goal, a ligand is binding to a pocket, which is located rim-exposed at the interface of the interacting proteins, for example as the phytotoxin Fusicoccin, which stabilizes the interaction of plant H+-ATPase and 14-3-3 protein by nearly a factor of 100. To suggest alternative leads, we performed a virtual screening campaign to discover new molecules putatively stabilizing this complex. Furthermore, we screen a dataset of 198 transient recognition protein-protein complexes for cavities, which are located rim-exposed at their interfaces. We provide evidence for high similarity between such rim-exposed cavities and usual ligands accommodating active sites of enzymes. This analysis suggests that rim-exposed cavities at protein-protein interfaces are druggable binding sites. Therefore, the principle of stabilizing protein-protein interactions seems to be a promising alternative to the approach of the competitive inhibition of such interactions by small molecules.