Interaction interfaces of protein domains are not topologically equivalent across families within superfamilies: Implications for metabolic and signaling pathways

Using a data set of aligned protein domain superfamilies of known three-dimensional structure, we compared the location of interdomain interfaces on the tertiary folds between members of distantly related protein domain superfamilies. The data set analyzed is comprised of interdomain interfaces, with domains occurring within a polypeptide chain and those between two polypeptide chains. We observe that, in general, the interfaces between protein domains are formed entirely in different locations on the tertiary folds in such pairs. This variation in the location of interface happens in protein domains involved in a wide range of functions, such as enzymes, adapters, and domains that bind protein ligands, or cofactors. While basic biochemical functionality is preserved at the domain superfamily level, the effect of biochemical function on protein assemblies is different in these protein domains related by superfamily. The divergence between proteins, in most cases, is coupled with domain recruitment, with different modes of interaction with the recruited domain. This is in complete contrast to the observation that in closely related homologous protein domains, almost always the interaction interfaces are topologically equivalent. In a small subset of interacting domains within proteins related by remote homology, we observe that the relative positioning of domains with respect to one another is preserved. Based on the analysis of multidomain proteins of known or unknown structure, we suggest that variation in protein-protein interactions in members within a superfamily could serve as diverging points in otherwise parallel metabolic or signaling pathways. We discuss a few representative cases of diverging pathways involving domains in a superfamily.     

Linking up and interacting with BRCT domains

BRCT domains are present in an ever expanding family of proteins that includes many DNA repair and checkpoint proteins. The most prominent member of the BRCT family is BRCA1, mutations in which are responsible for a high proportion of breast and ovarian cancers. BRCT domains act as protein–protein interaction modules and facilitate the formation of hetero- and homo-oligomers. The domains occur either singly or in pairs, with up to eight domains in a single protein. When in pairs the domains are separated by a short inter-BRCT linker. Numerous crystal structures have been determined for BRCT domains from a range of different proteins, which indicate that the overall structure of the BRCT domains is generally well conserved. In contrast, the positions and structures of the linker regions are more varied, as are the roles of the linkers. Here, we describe the protein–protein interactions involving three different inter-BRCT linker regions, those of DNA ligase IV (LigIV), Schizosaccharomyces pombe Crb2 and human 53BP1.     

Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains. This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families. At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data. The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families. We observe that almost half of all known families engage in interactions with domains from their own family. We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions. This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons. Copyright 12001 Academic Press.     

Improving the performance of DomainParser for structural domain partition using neural network

Structural domains are considered as the basic units of protein folding, evolution, function and design. Automatic decomposition of protein structures into structural domains, though after many years of investigation, remains a challenging and unsolved problem. Manual inspection still plays a key role in domain decomposition of a protein structure. We have previously developed a computer program, DomainParser, using network flow algorithms. The algorithm partitions a protein structure into domains accurately when the number of domains to be partitioned is known. However the performance drops when this number is unclear (the overall performance is 74.5% over a set of 1317 protein chains). Through utilization of various types of structural information including hydrophobic moment profile, we have developed an effective method for assessing the most probable number of domains a structure may have. The core of this method is a neural network, which is trained to discriminate correctly partitioned domains from incorrectly partitioned domains. When compared with the manual decomposition results given in the SCOP database, our new algorithm achieves higher decomposition accuracy (81.9%) on the same data set.     

Genome-wide functional annotation of dual-specificity protein- and lipid-binding modules that regulate protein interactions

Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that ~40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane-binding properties of 2,000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipid-binding PDZ domains into three classes based on the interplay between membrane- and protein-binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin 2, suggest that all classes of lipid-binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions.     

WW and SH3 domains, two different scaffolds to recognize proline-rich ligands

WW domains are small protein modules composed of approximately 40 amino acids. These domains fold as a stable, triple stranded beta-sheet and recognize proline-containing ligands. WW domains are found in many different signaling and structural proteins, often localized in the cytoplasm as well as in the cell nucleus. Based on analyses of seven structures of WW domains, we discuss their diverse binding preferences and sequence conservation patterns. While modeling WW domains for which structures have not been determined we uncovered a case of potential molecular and functional convergence between WW and SH3 domains. The binding surface of the modeled WW domain of Npw38 protein shows a remarkable similarity to the SH3 domain of Sem5 protein, confirming biochemical data on similar binding predilections of both domains.     

Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.     

The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro.

hic-5 protein is a member of the LIM protein family, containing four LIM domains in its C-terminal region. It is mainly localized in focal adhesions and shows striking similarity to paxillin in its LIM domains, although the function of these LIM domains has remained elusive. In the present study, we found that full-length and the C-terminal half of hic-5 protein, including four LIM domains, bound to DNA in a zinc-dependent manner in vitro . Mouse genomic fragments that specifically bound to the hic-5 protein were isolated by successive rounds of hic-5 protein-DNA complex immunoprecipitation and PCR amplification. Seven independent clones were isolated, which contained high amounts of G+A and/or a long A/T tract. A DNA binding protein blot assay revealed the specificity of the interaction between hic-5 protein and the DNA fragment. Using a series of truncated forms of the hic-5 LIM domains, each of the four LIM domains was found to contribute to DNA binding in a distinctive manner.     

Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule

Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20–25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.     

Distribution and function of new bacterial intein-like protein domains

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.     

Structure, function and evolution of multidomain proteins

Proteins are composed of evolutionary units called domains; the majority of proteins consist of at least two domains. These domains and nature of their interactions determine the function of the protein. The roles that combinations of domains play in the formation of the protein repertoire have been found by analysis of domain assignments to genome sequences. Additional findings on the geometry of domains have been gained from examination of three-dimensional protein structures. Future work will require a domain-centric functional classification scheme and efforts to determine structures of domain combinations.     

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.     

The multiple‐specificity landscape of modular peptide recognition domains

Modular protein interaction domains form the building blocks of eukaryotic signaling pathways. Many of them, known as peptide recognition domains, mediate protein interactions by recognizing short, linear amino acid stretches on the surface of their cognate partners with high specificity. Residues in these stretches are usually assumed to contribute independently to binding, which has led to a simplified understanding of protein interactions. Conversely, we observe in large binding peptide data sets that different residue positions display highly significant correlations for many domains in three distinct families (PDZ, SH3 and WW). These correlation patterns reveal a widespread occurrence of multiple binding specificities and give novel structural insights into protein interactions. For example, we predict a new binding mode of PDZ domains and structurally rationalize it for DLG1 PDZ1. We show that multiple specificity more accurately predicts protein interactions and experimentally validate some of the predictions for the human proteins DLG1 and SCRIB. Overall, our results reveal a rich specificity landscape in peptide recognition domains, suggesting new ways of encoding specificity in protein interaction networks.     

Structure-templated predictions of novel protein interactions from sequence information

The multitude of functions performed in the cell are largely controlled by a set of carefully orchestrated protein interactions often facilitated by specific binding of conserved domains in the interacting proteins. Interacting domains commonly exhibit distinct binding specificity to short and conserved recognition peptides called binding profiles. Although many conserved domains are known in nature, only a few have well-characterized binding profiles. Here, we describe a novel predictive method known as domain–motif interactions from structural topology (D-MIST) for elucidating the binding profiles of interacting domains. A set of domains and their corresponding binding profiles were derived from extant protein structures and protein interaction data and then used to predict novel protein interactions in yeast. A number of the predicted interactions were verified experimentally, including new interactions of the mitotic exit network, RNA polymerases, nucleotide metabolism enzymes, and the chaperone complex. These results demonstrate that new protein interactions can be predicted exclusively from sequence information.     

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3453 nucleotides and encodes a protein of 1151 amino acids with a molecular mass of 126408 Da. The deduced amino acid sequence of CBPA contained one domain highly similar to a catalytic domain of glycosyl hydrolases belonging to family 9, two linker-like domains and four domains of unknown function. Among the four domains of unknown function, the domains 1 and 2 region had significant homology in amino acid sequence with the cellulose-binding domains in the family 9 glycosyl hydrolases. The cloned gene was inserted into an expression vector, pBAD-TOPO, and expressed in Escherichia coli as a fused protein. The fused protein was detected by immunoblotting using antiserum against CBPA.     

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

An automatic method involving cluster analysis of secondary structures for the identification of domains in proteins.

With a growing number of structures available in the Brookhaven Protein Data Bank, automatic methods for domain identification are required for the construction of databases. Domains are considered to be clusters of secondary structure elements. Thus, helices and strands are first clustered using intersecondary structural distances between C alpha positions, and dendrograms based on this distance measure are used to identify domains. Individual domains are recognized by a disjoint factor, which enables the automatic identification and classification into disjoint, interacting, and conjoint domains. Application to a database of 83 protein families and 18 unique structures shows that the approach provides an effective delineation of boundaries and identifies those proteins that can be considered as a single domain. A quantitative estimate of the interaction between domains has been proposed. The database of protein domains is a useful tool for understanding protein folding, for recognizing protein folds, and for understanding structure-activity relationships.