A CD2-based model of yeast alpha-agglutinin elucidates solution properties and binding characteristics

We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains. Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall. Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin. In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins. Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains. Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin. The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation.     

Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type. On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible. We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.     

Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.

We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid. Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide. Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously. Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor. Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon. The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence. Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells. An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody. In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein. These results indicate that AG alpha 1 encodes alpha-agglutinin. Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor.     

Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit

BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of approximately 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an alpha-helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal alpha-helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional (1)H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.     

Residues within a conserved amino acid motif of domains 1 and 4 of VCAM- 1 are required for binding to VLA-4

Vascular cell adhesion molecule 1 (VCAM-1), a member of the Ig superfamily originally identified on activated endothelium, binds to the integrin very late antigen-4 (VLA-4), also known as alpha 4 beta 1 or CD49d/CD29, to support cell-cell adhesion. Studies based on cell adhesion to two alternatively spliced forms of VCAM-1 or to chimeric molecules generated from them and intercellular adhesion molecule-1 (ICAM-1) have demonstrated two VLA-4 binding sites on the predominate form of VCAM-1. Here, we studied VLA-4-dependent adhesion of the lymphoid tumor cell line Ramos to cells expressing wild type and mutant forms of VCAM-1. Results based on domain deletion mutants demonstrated the existence and independence of two VLA-4-binding sites located in the first and fourth domains of VCAM-1. Results based on amino acid substitution mutants demonstrated that residues within a linear sequence of six amino acids found in both domain 1 and 4 were required for VLA-4 binding to either domain. Five of these amino acids represent a conserved motif also found in ICAM domains. We propose that integrin binding to these Ig-like domains depends on residues within this conserved motif. Specificity of integrin binding to Ig-like domains may be regulated by a set of nonconserved residues distinct from the conserved motif.     

Design, engineering and production of functional single-chain T cell receptor ligands.

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen presenting cells (APCs) that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. The peptide binding/T cell recognition domains of rat MHC class II (alpha-1 and beta-1 domains) were expressed as a single exon for structural and functional characterization. These recombinant single-chain T cell receptor ligands (termed 'beta1alpha1' molecules) of approximately 200 amino acid residues were designed using the structural backbone of MHC class II molecules as template, and have been produced in Escherichia coli with and without N-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native MHC class II heterodimer. The proteins exhibited a cooperative two-state thermal folding-unfolding transition. Beta1alpha1 molecules with a covalently linked MBP-72-89 peptide showed increased stability to thermal unfolding relative to the empty beta1alpha1 molecules. This new class of small soluble polypeptide provides a template for designing and refining human homologues useful in detecting and regulating pathogenic T cells.     

Alpha-agglutinin expression in Saccharomyces cerevisiae

A polyclonal antiserum raised against purified alpha-agglutinin was made specific for alpha-agglutinin after adsorption with a cells. The adsorbed antiserum identified alpha-agglutinin peptides on Western blots and bound to cell surface alpha-agglutinin, inhibiting the binding of alpha cells to a cells. Using the antibody, we have determined that 1) the surface distribution of alpha-agglutinin on alpha cells is polar, 2) about 5 x 10(4) molecules/cell are constitutively expressed on strain X2180-1B (alpha) cells, and 3) treatment of alpha cells with the sex pheromone a-factor causes an increase in cell surface alpha-agglutinin, consistent with the a-factor induced increase in cell agglutinability.     

The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) alpha subunit. Cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B

Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87% identity at the amino acid level) and to the rest of the integrin alpha subunits (38%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or "L" domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.     

Streptococcal-host interactions. Structural and functional analysis of a Streptococcus sanguis receptor for a human salivary glycoprotein

Colonization of oral tissues by Streptococcus sanguis may be influenced by a mucin-like salivary glycoprotein (SAG) through a calcium-dependent interaction with a specific bacterial receptor. We report the nucleotide and deduced amino acid sequence of the S. sanguis receptor (SSP-5) and show that this protein may bind sialic acid residues of SAG. The SSP-5 protein contains three unique structural domains, two of which consist of repetitive amino acid sequences. The N-terminal domain is comprised of four tandem copies of an 82-residue repeat which exhibits homology to M protein of Streptococcus pyogenes. This region is highly charged and predicted to be alpha-helical. A second hydrophilic repetitive domain consists of three copies of a 39-amino acid sequence containing 30% proline flanked by nonrepetitive proline-rich sequence. The third domain consists of 48% proline and resides near the C terminus of the protein. Secondary structure analysis of the SSP-5 sequence also identified four potential helix-turn-helix motifs that resembled E-F hand calcium binding domains. The SSP-5 protein is highly homologous to a surface antigen expressed by the mutans streptococci and the domain structure of SSP-5 is conserved within this family of proteins. The interactions of SSP-5 and of intact S. sanguis with SAG were inhibited by neuraminidase digestion of the salivary glycoprotein and by simple sugars containing sialic acid, suggesting that sialic acid is the primary ligand involved in the binding reaction.     

Crystal structure of the human GGA1 GAT domain

GGAs are a family of vesicle-coating regulatory proteins that function in intracellular protein transport. A GGA molecule contains four domains, each mediating interaction with other proteins in carrying out intracellular transport. The GAT domain of GGAs has been identified as the structural entity that binds membrane-bound ARF, a molecular switch regulating vesicle-coat assembly. It also directly interacts with rabaptin5, an essential component of endosome fusion. A 2.8 A resolution crystal structure of the human GGA1 GAT domain is reported here. The GAT domain contains four helices and has an elongated shape with the longest dimension exceeding 80 A. Its longest helix is involved in two structural motifs: an N-terminal helix-loop-helix motif and a C-terminal three-helix bundle. The N-terminal motif harbors the most conservative amino acid sequence in the GGA GAT domains. Within this conserved region, a cluster of residues previously implicated in ARF binding forms a hydrophobic surface patch, which is likely to be the ARF-binding site. In addition, a structure-based mutagenesis-biochemical analysis demonstrates that the C-terminal three-helix bundle of this GAT domain is responsible for the rabaptin5 binding. These structural characteristics are consistent with a model supporting multiple functional roles for the GAT domain.     

An endogenous Drosophila receptor for glycans bearing alpha 1,3-linked core fucose residues

The genome of Drosophila melanogaster encodes several proteins that are predicted to contain Ca(2+)-dependent, C-type carbohydrate-recognition domains. The CG2958 gene encodes a protein containing 359 amino acid residues. Analysis of the CG2958 sequence suggests that it consists of an N-terminal domain found in other Drosophila proteins, a middle segment that is unique, and a C-terminal C-type carbohydrate-recognition domain. Expression studies show that the full-length protein is a tetramer formed by noncovalent association of disulfide-linked dimers that are linked through cysteine residues in the N-terminal domain. The expressed protein binds to immobilized yeast invertase through the C-terminal carbohydrate-recognition domain. Competition binding studies using monosaccharides demonstrate that CG2958 interacts specifically with fucose and mannose. Fucose binds approximately 5-fold better than mannose. Blotting studies reveal that the best glycoprotein ligands are those that contain N-linked glycans bearing alpha1,3-linked fucose residues. Binding is enhanced by the additional presence of alpha1,6-linked fucose. It has previously been proposed that labeling of the Drosophila neural system by anti-horseradish peroxidase antibodies is a result of the presence of difucosylated N-linked glycans. CG2958 is a potential endogenous receptor for such neural-specific carbohydrate epitopes.     

Orthologs in Arabidopsis thaliana of the Hsp70 interacting protein Hip

The Hsp70-interacting protein Hip binds to the adenosine triphosphatase domain of Hsp70, stabilizing it in the adenosine 5'-diphosphate-ligated conformation and promoting binding of target polypeptides. In mammalian cells, Hip is a component of the cytoplasmic chaperone heterocomplex that regulates signal transduction via interaction with hormone receptors and protein kinases. Analysis of the complete genome sequence of the model flowering plant Arabidopsis thaliana revealed 2 genes encoding Hip orthologs. The deduced sequence of AtHip-1 consists of 441 amino acid residues and is 42% identical to human Hip. AtHip-1 contains the same functional domains characterized in mammalian Hip, including an N-terminal dimerization domain, an acidic domain, 3 tetratricopeptide repeats flanked by a highly charged region, a series of degenerate GGMP repeats, and a C-terminal region similar to the Sti1/Hop/p60 protein. The deduced amino acid sequence of AtHip-2 consists of 380 amino acid residues. AtHip-2 consists of a truncated Hip-like domain that is 46% identical to human Hip, followed by a C-terminal domain related to thioredoxin. AtHip-2 is 63% identical to another Hip-thioredoxin protein recently identified in Vitis labrusca (grape). The truncated Hip domain in AtHip-2 includes the amino terminus, the acidic domain, and tetratricopeptide repeats with flanking charged region. Analyses of expressed sequence tag databases indicate that both AtHip-1 and AtHip-2 are expressed in A thaliana and that orthologs of Hip are also expressed widely in other plants. The similarity between AtHip-1 and its mammalian orthologs is consistent with a similar role in plant cells. The sequence of AtHip-2 suggests the possibility of additional unique chaperone functions.     

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.     

Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin

We used chicken alpha spectrin as a ligand probe to isolate Drosophila beta spectrin cDNA sequences from a lambda gt11 expression library. Analysis of 800 residues of deduced amino acid sequence at the amino- terminal end revealed a strikingly conserved domain of integral of 230 residues that shows a high degree of sequence similarity to the amino- terminal domains of alpha actinin and dystrophin. This conserved domain constitutes a new diagnostic criterion for spectrin-related proteins and allows the known properties of one of these proteins to predict functional properties of the others. The conservation of the amino- terminal domain, and other regions in spectrin, alpha actinin, and dystrophin, demonstrates that a common set of domains were linked in different combinations through evolution to generate the distinctive members of the spectrin superfamily.