RUN domains: a new family of domains involved in Ras-like GTPase signaling.

RUN domains are present in several proteins that are linked particularly to the functions of GTPases in the Rap and Rab families. They could hence play an important role in multiple Ras-like GTPase signaling pathways.

Hydrophobic cluster analysis and modeling of the human Rh protein three-dimensional structures.

Rh (Rhesus) is a major blood group system in man, which is clinically significant in transfusion medicine. Rh antigens are carried by an oligomer of two major erythroid specific polypeptides, the Rh (D and CcEe) proteins and the RhAG glycoprotein, that shared a common predicted structure with 12 transmembrane a-helices (M0 to M11). Non erythroid homologues of these proteins have been identified (RhBG and RhCG), notably in diverse organs specialized in ammonia production and excretion, such as kidney, liver and intestine. Phylogenetic studies and experimental evidence have shown that these proteins belong to the Amt/Mep/Rh protein superfamily of ammonium/methylammonium permease, but another view suggests that Rh proteins might function as CO2 gas channels. Until recently no information on the structure of these proteins were available. However, in the last two years, new insight has been gained into the structural features of Rh proteins (through the determination of the crystal structures of bacterial AmtB and archeaebacterial Amt-1. Here, models of the subunit and oligomeric architecture of human Rh proteins are proposed, based on a refined alignment with and crystal structure of the bacterial ammonia transporter AmtB, a member of the Amt/Mep/Rh superfamily. This alignment was performed considering invariant structural features, which were revealed through Hydrophobic Cluster Analysis, and led to propose alternative predictions for the less conserved regions, particularly in the N-terminal sequences. The Rh models, on which an additional Rh-specific, N-terminal helix M0 was tentatively positioned, were further assessed through the consideration of biochemical and immunochemical data, as well as of stereochemical and topological constraints. These models highlighted some Rh specific features that have not yet been reported. Among these, are the prediction of some critical residues, which may play a role in the channel function, but also in the stability of the subunit structure and oligomeric assembly. These results provide a basis to further understand the structure/function relationships of Rh proteins, and the alterations occurring in variant phenotypes.

Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.

Conformational study of the chromosomal protein MC1 from the archaebacterium Methanosarcina barkeri.

Methanogen chromosomal protein MC1 is a polypeptide of 93 amino acid residues (Mr 10,757) which represents the major protein associated with the DNA of the archaebacterium Methanosarcina barkeri and can protect DNA against thermal denaturation. The conformation of protein MC1 has been investigated by means of predictive methods, infrared spectroscopy, circular dichroism and tryptophan fluorescence studies. Protein MC1 has a low amount of alpha-helix but contains antiparallel beta-sheet strands. The larger hydrophobic cluster which contains tryptophan at position 61 appears buried in the protein. Addition of salts induces the unfolding of the protein and makes the tryptophan indole ring more rigid. With respect to its primary structure and its conformation, protein MC1 appears radically different from the chromosomal DNA-binding protein II (also called HU-type protein) in eubacteria.

A global taxonomy of loops in globular proteins.

A bank of loops from three to eight amino acid residues long has been constituted. On the basis of statistical analysis of occurrences of conformations and residue, loops could be divided into two parts: the side residues directly bonded to the secondary structure flanking element, and the inner part. The conformations of the side residues are correlated to the nature of their neighboring flanks, while the inner residues adopt conformations uncorrelated from one residue to the next; thus they are unrelated to the flanks. Two zones in the Ramachandran plot are important: alpha L and beta P. In particular, the high occurrence of alpha L, mainly occupied by glycine residues, is necessary to induce flexibility and thus allow loops to comply with the geometrical constraints of the flanks. An algorithm of clustering has been used to aggregate loops of the same length within families of similar 3D structures. At each position in each cluster, sequence and conformational signatures have been deduced if the occurrence of a residue (or a conformation) is higher than an equiprobable distribution over all clusters. The result is that some positions favor particular amino acids and conformations, which are typical of a cluster although not unique. This is an indication of a relation between structure and sequence in loops. A taxonomy is proposed that classifies the various clusters. It relies on two terms: the mean distance between the first and last C alpha in one cluster and, perpendicular to this line, the distance to the center of gravity of the cluster. It is noteworthy that the differently populated clusters represented in such 2D plots can be separated. Thus, although the conformations of loops in globular proteins could cover a continuum, it has been possible to cluster them into a limited number of well populated families and superfamilies. This basic feature of protein architecture could be further exploited to better predict their geometry.

New efficient statistical sequence-dependent structure prediction of short to medium-sized protein loops based on an exhaustive loop classification.

A bank of 13,563 loops from three to eight amino acid residues long, representing motifs between two consecutive regular secondary structures, has been derived from protein structures presenting less than 95 % sequence identity. Statistical analyses of occurrences of conformations and residues revealed length-dependent over-representations of particular amino acids (glycine, proline, asparagine, serine, and aspartate) and conformations (alphaL, epsilon, betaPregions of the Ramachandran plot). A position-dependent distribution of these occurrences was observed for N and C-terminal residues, which are correlated to the nature of the flanking regions. Loops of the same length were clustered into statistically meaningful families on the basis of their backbone structures when placed in a common reference frame, independent of the flanks. These clusters present significantly different distributions of sequence, conformations, and endpoint residue Calphadistances. On the basis of the sequence-structure correlation of this clustering, an automatic loop modeling algorithm was developed. Based on the knowledge of its sequence and of its flank backbone structures each query loop is assigned to a family and target loop supports are selected in this family. The support backbones of these target loops are then adjusted on flanking structures by partial exploration of the conformational space. Loop closure is performed by energy minimization for each support and the final model is chosen among connected supports based upon energy criteria. The quality of the prediction is evaluated by the root-mean-square deviation (rmsd) between the final model and the native loops when the whole bank is re-attributed on itself with a Jackknife test. This average rmsd ranges from 1.1 A for three-residue loops to 3.8 A for eight-residue loops. A few poorly predicted loops are inescapable, considering the high level of diversity in loops and the lack of environment data. To overcome such modeling problems, a statistical reliability score was assigned for each prediction. This score is correlated to the quality of the prediction, in terms of rmsd, and thus improves the selection accuracy of the model. The algorithm efficiency was compared to CASP3 target loop predictions. Moreover, when tested on a test loop bank, this algorithm was shown to be robust when the loops are not precisely delimited, therefore proving to be a useful tool in practice for protein modeling.

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family.

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.

Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor.

Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four small nuclear ribonucleoprotein particles (snRNPs) and additional protein factors. The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the first step of splicing. In this work, we used the in vivo yeast two-hybrid system and in vitro immunoprecipitation assays to show that yPrp4 interacts with yPrp3, another U4/U6 snRNP protein. To investigate the domain of yPrp4 that directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4 and point mutations in the C-terminal half of the molecule, and we tested the resulting prp4 mutants for cell viability and for their ability to interact with yPrp3. We could not define any particular sequence in the first 161 amino acid residues that are specifically required for protein-protein interactions. However, deletion of a small basic-rich region of 30 amino acid residues is lethal to the cells. Analysis of the C terminus prp4 mutants obtained clearly shows that this region of yPrp4 represents the primary domain of interaction with yPrp3. Interestingly, yPrp4 shows significant similarity in its C-terminal half to the beta-subunits of G proteins. We have generated a three-dimensional computer model of this domain, consisting of a seven-bladed beta-propeller based on the crystalline structure of beta-transducin. Several lines of evidence suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by the long variable loops linking the beta-strands of the propeller. This surface could be used as a scaffold for generating an RNA-protein complex.

Crystallization and preliminary X-ray diffraction results of trichorzianine A 1, a peptide with nineteen residues from Trichoderma harzianum.

Trichorzianine A 1 is one of the main components of a mixture of related antibiotic peptides (trichorzianines) produced by the fungus Trichoderma harzianum. Good crystals were obtained and allowed X-ray diffraction up to 0.8 A resolution. The space group is orthorhombic, C222(1), Z = 8, a = 64.8 (1) A, b = 9.33 (3) A, c = 39.9 (1) A. The solvent content is only 12%, preventing a heavy ion diffusion. So, we are trying to obtain the structure by direct methods.

Crystallization and preliminary X-ray diffraction studies of a protective cloned 28 kDa glutathione S-transferase from Schistosoma mansoni.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.

The bovine leukemia virus (BLV) envelope glycoprotein gp51 as a general model for the design of a subunit vaccine against retroviral infection: mapping of functional sites through immunological and structural data.

Further advances in retroviral vaccine development require a better understanding of the antigenic structure of the envelope complex which is directly involved in infectivity events such as receptor recognition and membrane fusion. To design an optimal vaccine against BLV infection, we chose an approach based on the use of synthetic peptides covering 78% of the gp51 sequence in order to select only those segments that could induce a protective response via cellular and humoral immunity. On the other hand, we built a model of the BLV env glycoprotein 3D organization, based upon the very sensitive hydrophobic cluster analysis (HCA). The major information highlighted from this model is that the two loops, against which the most efficient neutralizing antipeptides antibodies are directed against, are in close proximity at the top of the "head" and could represent a potential site for receptor binding. These two peptides are of particular interest since they induce also a helper T-cell response. We further propose that the BLV envelope glycoprotein oligomerizes as a trimer.

HYR, an extracellular module involved in cellular adhesion and related to the immunoglobulin-like fold.

Domains belonging to the immunoglobulin-like fold are responsible for a wide variety of molecular recognition processes. Here we describe a new family of domains, the HYR family, which is predicted to belong to this fold, and which appears to be involved in cellular adhesion. HYR domains were identified in several eukaryotic proteins, often associated with Complement Control Protein (CCP) modules or arranged in multiple copies. Our analysis provides a sequence and structural basis for understanding the role of these domains in interaction mechanisms and leads to further characterization of heretofore undescribed repeated domains with similar folds found in several bacterial proteins involved in enzymatic activities (some chitinases) or in cell surface adhesion (streptococcal C-alpha antigen).

Cellulase families revealed by hydrophobic cluster analysis.

The amino acid sequences of 21 beta-glycanases have been compared by hydrophobic cluster analysis. Six families of cellulases have been identified on the basis of primary structure homology: (A) endoglucanases B, C and E of Clostridium thermocellum; endoglucanases of Erwinia chrysanthemi and Bacillus sp.; endoglucanase III of Trichoderma reesei; endoglucanase I of Schizophyllum commune; (B) cellobiohydrolase II of T. reesei; endoglucanases of Cellulomonas fimi and Streptomyces sp; (C) cellobiohydrolases I of T. reesei and of Phanerochaete chrysosporium; endoglucanase I of T. reesei; (D) endoglucanase A of C. thermocellum and an endoglucanase from Ce. uda; (E) endoglucanase D of C. thermocellum and an endoglucanase from Pseudomonas fluorescens; (F) xylanases of C. thermocellum and of Cryptococcus albidus and the cellobio-hydrolase of Ce. fimi. For each family, conserved potentially catalytic residues have have been listed and previous allocations of the active-site residues are evaluated in the light of the alignment of the amino acid sequences. A strong homology is also reported for the putative cellulose-binding domains of cellulases of Ce. fimi and of P. fluorescens.

From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair.

Inherited mutations in BRCA1 predispose to breast and ovarian cancer, but the biological function of the BRCA1 protein has remained largely elusive. The recent correspondence of Koonin et al. [Koonin, E.V., Altschul, S.F. and Bork, P. (1996) Nature Genet. 13, 266-267] has emphasized the potential importance of the BRCA1 C-terminal region for BRCA1-mediated breast cancer suppression, as this domain shows similarities with the C-terminal regions of a p53-binding protein (53BP1), the yeast RAD9 protein involved in DNA repair, and two uncharacterized, hypothetical proteins (KIAA0170 and SPAC19G10.7). The highlighted domain has been suggested to be the result of an internal duplication, each of the tandem domains being designated as a 'BRCT domain' (for BRCA1 C-terminus). Sequence analysis using hydrophobic cluster analysis reveals here the presence of 50 copies of the BRCT domain in 23 different proteins, including, in addition to BRCA1, 53BP1 and RAD9, XRCC1, RAD4, Ect2, REV1, Crb2, RAP1, terminal deoxynucleotidyltransferases (TdT) and three eukaryotic DNA ligases. Most of these proteins are known to be involved in DNA repair. The BRCT domain is not limited to the C-termini of protein sequences and can be found in multiple copies or in a single copy as in RAP1 and TdT, suggesting that it could well constitute an autonomous folding unit of approx. 90-100 amino acids.

Predicting the protein folding nucleus from sequences [correction of a sequence].

Understanding the mechanism of protein folding would allow prediction of the three-dimensional structure from sequence data alone. It has been shown that small proteins fold in a small number of kinetic steps and that significantly populated intermediate states exist for some of them. Studies of these intermediates have demonstrated the existence of specific interactions established during the initial stages of folding. Comparison of the amino acids participating in these specific and essential interactions and constituting the folding nucleus with conserved hydrophobic positions of a given fold shows a striking correspondence. This finding opens the perspective of predicting the folding nucleus knowing only a set of divergent sequences of a protein family.

Trigger factor, one of the Escherichia coli chaperone proteins, is an original member of the FKBP family.

The trigger factor of Escherichia coli is known as a chaperone protein which forms soluble complexes with the precursor to outer membrane protein A and assists in the maintenance of translocation competence. Sequence analysis shows that trigger factor contains a domain belonging to the FK506-binding protein (FKBP) family and possessing all the amino acids necessary for FK506 binding and peptidyl-prolyl cis-trans isomerase (Ppiase) activity. Consequently, this protein could be directly involved in the unfolding/folding processes occurring during translocation across the E. coli plasma membrane and, more generally, in facilitating protein folding. The central position of the FKBP domain within the trigger factor sequence as well as several original features of the loops surrounding the FK506-binding pocket are not found in any other FKBPs, making it undetectable by the Fkbp-Ppiase signature patterns.

The BAH (bromo-adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation.

Using sensitive methods of sequence analysis including hydrophobic cluster analysis, we report here a hitherto undescribed family of modules, the BAH (bromo-adjacent homology) family, which includes proteins such as eukaryotic DNA (cytosine-5) methyltransferases, the origin recognition complex 1 (Orc1) proteins, as well as several proteins involved in transcriptional regulation. The BAH domain appears to act as a protein-protein interaction module specialized in gene silencing, as suggested for example by its interaction within yeast Orc1p with the silent information regulator Sir1p. The BAH module might therefore play an important role by linking DNA methylation, replication and transcriptional regulation.

Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences.

A new method for comparing and aligning protein sequences is described. This method, hydrophobic cluster analysis (HCA), relies upon a two-dimensional (2D) representation of the sequences. Hydrophobic clusters are determined in this 2D pattern and then used for the sequence comparisons. The method does not require powerful computer resources and can deal with distantly related proteins, even if no 3D data are available. This is illustrated in the present report by a comparison of human haemoglobin with leghaemoglobin, a comparison of the two domains of liver rhodanese (thiosulphate sulphurtransferase) and a comparison of plastocyanin and azurin.

Sequence and 3D structural relationships between mammalian Ras- and Rho-specific GTPase-activating proteins (GAPs): the cradle fold.

An extensive study of both sequence and recent 3D structural data concerning GTPase interacting domains of Ras- and Rho-specific GTPase-activating proteins (GAPs) shows that these two subfamilies share a same 3D scaffold and are thus related to each other. This relationship has heretofore remained undetected although these domains of similar size are both totally alpha-helical and activate nearly structurally identical targets (Ras and Rho proteins). In this report, sequence similarities correlated to 3D structures of p120rasGAP and p50rhoGAP were detected using the sensitive two-dimensional method hydrophobic cluster analysis (HCA). These patterns were further extended to other members in each subfamily and the geometry orientation of crucial arginines R789 in p120 and R282 in p50 and of important stabilizing residues like p120R903 and p50N391 was confirmed. This overall structural relationship is centered on an invariant motif of three consecutive helices that we suggest to name the 'cradle fold'. This observation opens new perspectives to understand how small GTPases are specifically regulated.