László Bányai

COMMON EVOLUTIONARY ORIGIN OF THE FIBRIN-BINDING STRUCTURES OF FIBRONECTIN AND TISSUE-TYPE PLASMINOGEN ACTIVATOR

Comparison of the primary structures of high‐M r urokinase and tissue‐type plasminogen activator reveals a high degree of structural homology between the two proteins, except that tissue activator contains a 43 residue long amino‐terminal region, which has no counterpart in urokinase. We show that this segment is homologous with the finger‐domains responsible for the fibrin‐affinity of fibronectin. Limited proteolysis of the amino‐terminal region of plasminogen activator was found to lead to a loss of the fibrin‐affinity of the enzyme. It is suggested that the finger‐domains of fibronectin and tissue‐types plasminogen activator have similar functions and that the finger‐domains of the two proteins evolved from a common ancestral fibrin‐binding domain.

Kringles: modules specialized for protein binding: Homology of the gelatin-binding region of fibronectin with the kringle structures of proteases

Prothrombin, plasminogen, urokinase‐ and tissue‐type plasminogen activators contain homologous structures known as kringles. The kringles correspond to autonomous structural and folding domains which mediate the binding of these multidomain proteins to other proteins. During evolution the different kringles retained the same gross architecture, the kringle‐fold, yet diverged to bind different proteins. We show that the amino acid sequences of the type II structures of the gelatin‐binding region of fibronectin are homologous with those of the protease‐kringles. Prediction of secondary structures revealed a remarkable agreement in the positions of predicted β‐sheets, suggesting that the folding of kringles and type II structures may also be similar. As a corollary of this finding, the disulphide‐bridge pattern of type II structures is shown to be homologous to that in kringles. It is noteworthy that protease‐kringles and fibronectin type II structures have similar functions inasmuch as they mediate the binding of multidomain proteins to other proteins. It is proposed that the kringles of proteases and type II structures of fibronectin evolved from a common ancestral protein binding module.

The PAN module: the N‐terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins

Based on homology search and structure prediction methods we show that (1) the N‐terminal N domains of members of the plasminogen/hepatocyte growth factor family, (2) the apple domains of the plasma prekallikrein/coagulation factor XI family, and (3) domains of various nematode proteins belong to the same module superfamily, hereafter referred to as the PAN module. The patterns of conserved residues correspond to secondary structural elements of the known three‐dimensional structure of hepatocyte growth factor N domain, therefore we predict a similar fold for all members of this superfamily. Based on available functional informations on apple domains and N domains, it is clear that PAN modules have significant functional versatility, they fulfill diverse biological functions by mediating protein‐protein or protein‐carbohydrate interactions.

Modules, multidomain proteins and organismic complexity

Originally the term ‘protein module’ was coined to distinguish mobile domains that frequently occur as building blocks of diverse multidomain proteins from ‘static’ domains that usually exist only as stand‐alone units of single‐domain proteins. Despite the widespread use of the term ‘mobile domain’, the distinction between static and mobile domains is rather vague as it is not easy to quantify the mobility of domains. In the present work we show that the most appropriate measure of the mobility of domains is the number of types of local environments in which a given domain is present. Ranking of domains with respect to this parameter in different evolutionary lineages highlighted marked differences in the propensity of domains to form multidomain proteins. Our analyses have also shown that there is a correlation between domain size and domain mobility: smaller domains are more likely to be used in the construction of multidomain proteins, whereas larger domains are more likely to be static, stand‐alone domains. It is also shown that shuffling of a limited set of modules was facilitated by intronic recombination in the metazoan lineage and this has contributed significantly to the emergence of novel complex multidomain proteins, novel functions and increased organismic complexity of metazoa.

Refined solution structure and ligand-binding properties of PDC-109 domain b. A collagen-binding type II domain.

We have determined, via 1H-n.m.r., the solution conformation of the collagen-binding b-domain of the bovine seminal fluid protein PDC-109 (PDC-109/b). The structure determination is based on 341 interproton distance estimates and 42 dihedral angle estimates: a set of 24 initial structures were computed; 12 using the variable target function program DIANA, and 12 using the metric matrix program DISGEO. These structures were optimized by restrained energy minimization and dynamic simulated annealing using the CHARMM and X-PLOR programs. The average pairwise root-mean-square difference (r.m.s.d) between the optimized DIANA (DISGEO) structures is 0.71 A (0.82 A) for the backbone atoms, and 1.73 A (2.03 A) for all atoms. Both sets of structures exhibit the same global fold, secondary structure and placement of most non-polar side-chains. Two central antiparallel beta-sheets, which lie roughly perpendicular to each other, and two irregular loops support a large, partially exposed, hydrophobic surface that defines a putative binding site. A test of a hybrid relaxation matrix-based distance refinement protocol (MIDGE program) was performed using a normalized 250 millisecond NOESY spectrum. The resulting distances were input to the molecular mechanics/dynamics procedures mentioned above in order to optimize the DIANA structures. Our results indicate that relaxation matrix refinement of distances is most useful when used conservatively for identifying underestimated distance constraints. 1H-n.m.r. monitored ligand titration experiments revealed definite, albeit weak, binding interactions for phenethylamine and leucine analogs (Ka less than or equal to 25 M-1). Residues perturbed by ligand binding include Tyr7, Trp26, Tyr33, Asp34 and Trp39. These results suggest that PDC-109/b may recognize specific leucine and/or isoleucine-containing sequences within collagen.

A human protein containing multiple types of protease-inhibitory modules

By using sensitive homology-search and gene-finding programs, we have found that a genomic region from the tip of the short arm of human chromosome 16 (16p13.3) encodes a putative secreted protein consisting of a domain related to the whey acidic protein (WAP) domain, a domain homologous with follistatin modules of the Kazal-domain family (FS module), an immunoglobulin-related domain (Ig domain), two tandem domains related to Kunitz-type protease inhibitor modules (KU domains), and a domain belonging to the recently defined NTR-module family (NTR domain). The gene encoding these WAP, FS, Ig, KU, and NTR modules (hereafter referred to as the WFIKKN gene) is intron-depleted—its single 1,157-bp intron splits the WAP module. The validity of our gene prediction was confirmed by sequencing a WFIKKN cDNA cloned from a lung cDNA library. Studies on the tissue-expression pattern of the WFIKKN gene have shown that the gene is expressed primarily in pancreas, kidney, liver, placenta, and lung. As to the function of the WFIKKN protein, it is noteworthy that it contains FS, WAP, and KU modules, i.e., three different module types homologous with domains frequently involved in inhibition of serine proteases. The protein also contains an NTR module, a domain type implicated in inhibition of zinc metalloproteinases of the metzincin family. On the basis of its intriguing homologies, we suggest that the WFIKKN protein is a multivalent protease inhibitor that may control the action of multiple types of serine proteases as well as metalloproteinase(s).

Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase.

The fibronectin‐related region of the 72 kDa type IV procollagenase has been expressed in E. coli as a β‐galactosidase fusion product. The fragment containing the three type II units of the protein was found to have affinity for denatured collagen, suggesting that these domains may be responsible for the collagen‐affinity of type IV collagenase. We have also shown that segment Ala‐Ala‐His‐Glu of type IV collagenase (residues 372–375), which is similar to a fibronectin‐segment previously implicated in collagen‐binding, is not essential for binding activity.

Amoebapore homologs of Caenorhabditis elegans

It is shown that the Caenorhabditis elegans genome contains several distantly related members of the gene family of saposin-like proteins. The putative products of genes T07C4.4, T08A9.7A, T08A9.7B, T08A9.8, T08A9.9, T08A9.10 are similar to the amoebapores of Entamoeba histolytica, granulysin of cytotoxic T lymphocytes and a putative amoebapore-related protein of the liver fluke Fasciola hepatica inasmuch as they consist of only a single saposin-like domain and a secretory signal peptide. The saposin-like domain of protein T07C4.4, which is most closely related to NK-lysin and granulysin, has been expressed in Escherichia coli and the recombinant protein was shown to have a circular dichroism spectrum consistent with the helix bundle structure characteristic of saposin-like domains. Recombinant T07C4.4 protein was found to have antibacterial activity, suggesting that these amoebapore homologs may play a role in antibacterial mechanisms of C. elegans.

Identification and correction of abnormal, incomplete and mispredicted proteins in public databases

BackgroundDespite significant improvements in computational annotation of genomes, sequences of abnormal, incomplete or incorrectly predicted genes and proteins remain abundant in public databases. Since the majority of incomplete, abnormal or mispredicted entries are not annotated as such, these errors seriously affect the reliability of these databases. Here we describe the MisPred approach that may provide an efficient means for the quality control of databases. The current version of the MisPred approach uses five distinct routines for identifying abnormal, incomplete or mispredicted entries based on the principle that a sequence is likely to be incorrect if some of its features conflict with our current knowledge about protein-coding genes and proteins: (i) conflict between the predicted subcellular localization of proteins and the absence of the corresponding sequence signals; (ii) presence of extracellular and cytoplasmic domains and the absence of transmembrane segments; (iii) co-occurrence of extracellular and nuclear domains; (iv) violation of domain integrity; (v) chimeras encoded by two or more genes located on different chromosomes.ResultsAnalyses of predicted EnsEMBL protein sequences of nine deuterostome (Homo sapiens, Mus musculus, Rattus norvegicus, Monodelphis domestica, Gallus gallus, Xenopus tropicalis, Fugu rubripes, Danio rerio and Ciona intestinalis) and two protostome species (Caenorhabditis elegans and Drosophila melanogaster) have revealed that the absence of expected signal peptides and violation of domain integrity account for the majority of mispredictions. Analyses of sequences predicted by NCBIs GNOMON annotation pipeline show that the rates of mispredictions are comparable to those of EnsEMBL. Interestingly, even the manually curated UniProtKB/Swiss-Prot dataset is contaminated with mispredicted or abnormal proteins, although to a much lesser extent than UniProtKB/TrEMBL or the EnsEMBL or GNOMON-predicted entries.ConclusionMisPred works efficiently in identifying errors in predictions generated by the most reliable gene prediction tools such as the EnsEMBL and NCBIs GNOMON pipelines and also guides the correction of errors. We suggest that application of the MisPred approach will significantly improve the quality of gene predictions and the associated databases.

NMR structure of the LCCL domain and implications for DFNA9 deafness disorder.

The LCCL domain is a recently discovered, conserved protein module named after its presence in Limulus factor C, cochlear protein Coch‐5b2 and late gestation lung protein Lgl1. The LCCL domain plays a key role in the autosomal dominant human deafness disorder DFNA9. Here we report the nuclear magnetic resonance (NMR) structure of the LCCL domain from human Coch‐5b2, where dominant mutations leading to DFNA9 deafness disorder have been identified. The fold is novel. Four of the five known DFNA9 mutations are shown to involve at least partially solvent‐exposed residues. Except for the Trp91Arg mutant, expression of these four LCCL mutants resulted in misfolded proteins. These results suggest that Trp91 participates in the interaction with a binding partner. The unexpected sensitivity of the fold with respect to mutations of solvent‐accessible residues might be attributed to interference with the folding pathway of this disulfide‐containing domain.