Domain structure and molecular flexibility of streptococcal M proteinIn Situ probed by limited proteolysis

Serologically distinct group A streptococcal M proteins, the antiphagocytic determinants of the bacteria, have a highly repetitive sequence and exhibit a heptad periodicity characteristic of alpha-helical coiled-coil proteins. Based on the differences in the pattern of heptad periodicity, the coiled-coil region of the complete M molecule has been divided into three distinct domains: I, II, and III. Domains I and II together constitute the variable part of M protein, whereas domain III is conserved among serotypes. Pepsin treatment of the M5, M6, and M24 streptococci results in a preferential cleavage of their M molecules between the predicted domains II and III, releasing biologically active fragments of the respective M proteins. Thus, a pepsin cleavage site at the junction of their variable and conserved regions is conserved in the M5, M6, and M24 proteins. In contrast, in the case of the M49 streptococci, the primary site of pepsin cleavage was observed to be within the conserved region of the M49 molecule, rather than at the junction of its variable and conserved regions. Despite containing part of the conserved region, the PepM49 protein is significantly smaller than the pepsin fragments of the M5, M6, and M24 proteins, which contain only the variable regions. However, in addition to the major PepM49 species, the pepsin digest of the type-49 streptococci also contained a smaller fragment, PepM49/a, as a minor component. Its formation was extremely sensitive to thepH of pepsin digestion. PepM49/a, which retains both the propensity to attain an alpha-helical conformation and the opsonic antibody epitope of the M49 molecule, contains only domains I and II like the other PepM proteins. Thus, as in the M5, M6, and M24 proteins, a pepsin cleavage site at the junction of the variable and conserved regions is indeed present in the M49 molecule, but is much less accessible relative to the other serotypes. Thus, the pepsin cleavage sites in the M protein correlate quite well with the boundaries of structurally distinct domains reflected by the predictive analysis. These sites apparently represent the flexible/hinge regions of the molecule. PepM49/a is the least repetitive and the shortest of the M protein pepsin fragments isolated so far. These results suggest that the flexibility of the interdomain regions in M protein may be dependent on the molecular size of their variable domains. The placement of a more accessible hinge within the conserved part of the M49 molecule, rather than at the junction of the variable and conserved domains, suggests that a critical molecular size may be essential for the efficient functioning of the M molecule.     

Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes

The complete amino acid sequence of PepM49, a peptic fragment of the group A streptococcal type 49 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment retains the opsonic antibody epitope of the native molecule. The sequence of PepM49, as determined by automated Edman degradations of the uncleaved molecule, and its tryptic and chymotryptic peptides, consists of a total of 143 residues (Mr = 17,187). PepM49, a nephritis-associated M protein serotype, exhibits significant internal homology in its sequence. However, identical sequence repeats of the kind seen in the rheumatic fever-associated serotypes M5, M6, and M24, are absent in PepM49. PepM49 exhibits varying degrees of homology with the M5, M6, and M24 proteins, which is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive analysis as well as CD measurements revealed a high propensity of the PepM49 molecule to assume an alpha-helical conformation. Furthermore, a heptad periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil proteins, extends over the entire length of the PepM49 protein. The differences in the nonpolar residue distribution divide the PepM49 sequence into three distinct domains, similar to those seen earlier in the M5 and M6 proteins. Together, these studies establish a conserved conformational design for the sequentially diverse M protein serotypes. However, the pattern of heptad periodicity in the PepM49 protein is quite distinct from that present in the PepM5 and M6 proteins, suggesting distinct differences in structural features among conformationally similar M protein serotypes. This may have relevance to the pathological differences associated with these M protein serotypes.     

Heptad motifs within the distal subdomain of the coiled-coil rod region of M protein from rheumatic fever and nephritis associated serotypes of group A streptococci are distinct from each other: Nucleotide sequence of the M57 gene and relation of the deduced amino acid sequence to other M proteins

Streptococcal M protein, a dimeric alpha helical coiled-coil molecule, is an antigenically variable virulence factor on the surface of the bacteria. Our recent conformational analysis of the complete sequence of the M6 protein led us to propose a basic model for the M protein consisting of an extended central coiled-coil rod domain flanked by a variable N-terminal and a conserved C-terminal end domains. The central coiled-coil rod domain of M protein, which constitutes the major part of the M molecule, is made up of repeating heptads of the generalized sequence a-b-c-d-e-f-g, wherein a and d are predominantly apolar residues. Based on the differences in the heptad pattern of apolar residues and internal sequence homology, the central coiled-coil rod domain of M protein could be further divided into three subdomains I, II, and III. The streptococcal sequelae rheumatic fever (RF) and acute glomerulonephritis (AGN) have been known to be associated with distinct serotypes. Consistent with this, we observed that the AGN associated M49 protein exhibits a heptad motif that is distinct from the RF associated M5 and M6 proteins. Asn and Leu predominated in the a and d positions, respectively, in subdomain I of the M5 and M6 proteins, whereas apolar residues predominated in both these positions in the M49 protein. To establish whether the heptad motif of M49 is unique to this protein, or is a general characteristic of nephritis-associated serotypes, the amino acid sequence of M57, another nephritis-associated serotype, has now been examined. The gene encoding M57 was amplified by PCR, cloned into pUC19 vector, and sequenced. The C-terminal half of M57 is highly homologous to other M proteins (conserved region). In contrast, its N-terminal half (variable region) revealed no significant homology with any of the M proteins. Heptad periodicity analysis of the M57 sequence revealed that the basic design principles, consisting of distinct domains observed in the M6 protein, are also conserved in the M57 molecule. However, the heptad motif within the coiled-coil subdomain I of M57 was distinct from M5 and M6 but similar to M49. Similar analyses of the heptad characteristics within the reported sequences of M1, M12, and M24 proteins further confirmed the conservation of the overall architectural design of sequentially distinct M proteins. Furthermore, the heptad motif within subdomain I of the AGN-associated serotypes M1 and M12 was similar to M49 and M57, whereas that of the RF associated M24 was similar to the M5 and M6 proteins. These results clearly demonstrate a correlation between the heptad motifs within the distal coiled-coil subdomain of the M proteins from different streptococcal serotypes and their epidemiological association with the sequelae AGN and RF.     

Conformational characteristics of the complete sequence of group A streptococcal M6 protein

M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were "core" nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.     

Domain interactions within Fzo1 oligomers are essential for mitochondrial fusion

Mitofusins are conserved GTPases essential for the fusion of mitochondria. These mitochondrial outer membrane proteins contain a GTPase domain and two or three regions with hydrophobic heptad repeats, but little is known about how these domains interact to mediate mitochondrial fusion. To address this issue, we have analyzed the yeast mitofusin Fzo1p and find that mutation of any of the three heptad repeat regions (HRN, HR1, and HR2) leads to a null allele. Specific pairs of null alleles show robust complementation, indicating that functional domains need not exist on the same molecule. Biochemical analysis indicates that this complementation is due to Fzo1p oligomerization mediated by multiple domain interactions. Moreover, we find that two non-overlapping protein fragments, one consisting of HRN/GTPase and the other consisting of HR1/HR2, can form a complex that reconstitutes Fzo1p fusion activity. Each of the null alleles disrupts the interaction of these two fragments, suggesting that we have identified a key interaction involving the GTPase domain and heptad repeats essential for fusion.     

The amino-terminal region of group A streptococcal M protein determines its molecular state of assembly and function

Group A streptococcal M protein, a major virulence factor, is an alpha-helical coiled-coil dimer on the surface of the bacteria. Limited proteolysis of type 57 streptococcus with pepsin released two fragments of the M57 molecule, with apparent molecular weights of 32,000 and 27,000 on SDS-PAGE. However, on gel filtration under nondenaturing conditions, each of these proteins eluted as two distinct molecular forms. The two forms corresponded to their dimeric and monomeric state as compared to the gel filtration characteristics of known dimeric coiled-coil proteins. The results of sedimentation equilibrium measurements were consistent with this, but further indicated that the dimeric form consisted of a dimer in rapid equilibrium with its monomer, whereas the monomeric form does not dimerize. The monomeric form was the predominant species for the 27 kD species, whereas the dimeric form predominated for the 32 kD species. Sequence analysis revealed the 27 kD species to be a truncated derivative of the 32 kD PepM57 species, lacking the N-terminal nonheptad region of the M57 molecule. These data strongly suggested that the N-terminal nonheptad region of PepM57 is important in determining the molecular state of the molecule. Consistent with this, PepM49, another nephritis-associated serotype, which lacks the nonheptad N-terminal region, also eluted as a monomer on gel filtration under nondenaturing conditions. Furthermore, removal of the N-terminal nonheptad segment of the dimeric PepM6 protein converted it into a monomeric form. The dimeric molecular form of both the 32 kD PepM57 and the 27 kD PepM57 did not represent a stable state of assembly, and were susceptible to conversion to the corresponding monomeric molecular forms by simple treatments, such as lyophilization. The 27 kD PepM57 exhibited a greater propensity than the 32 kD species to exist in the monomeric form. The 32 kD species contained the opsonic epitope of the M57 molecule, whereas the 27 kD species lacked the same. This is consistent with the previous reports on the importance of the N-terminal region of M protein for its opsonic activity. Together, these results strongly suggest that, in addition to its importance for the biological function, the N-terminal region of the M protein plays a dominant role in determining the molecular state of the M molecule, as well as its stability.     

Mapping the immunodeterminants of the complete streptococcal M6 protein molecule. Identification of an immunodominant region

The immune response to the complete streptococcal M6 protein was examined by kinetic ELISA to determine the reactivity of rabbit and human sera to M6 peptides representing 82% of the native molecule. The results revealed that rabbits immunized with purified native M6 protein or whole streptococci responded by reacting early and predominantly to one of the three sequence repeat regions of the molecule, the B-repeat, antibodies which have been shown to be non-opsonic. Antibodies to peptides representing the hypervariable N-terminal and adjacent A-repeat regions appear when opsonic antibodies are detected in the serum. Antibodies to peptides located within the conserved C-terminal half of the molecule (proximal to the cell) were restricted even after several immunizations. An examination of human sera from individuals with no recent streptococcal infection (greater than 3 yr), revealed that those sera opsonic for M6 streptococci contained antibodies reactive predominantly to the N-terminal and A-repeat regions, supporting the view that opsonic antibodies are long lived. Nonopsonic human sera to M6 streptococci exhibited a low reactivity to all peptides. However, by Western blot analysis, all human sera tested contained antibodies to the conserved region of the molecule, whereas only sera opsonic for M6 streptococci reacted with the variable region. Evidence is presented supporting the view that antibodies to the conserved regions of the M molecule may be conformation dependent.     

The role of disulfide bonds and alpha-helical coiled-coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins

Type XIII collagen is a type II transmembrane protein with three collagenous (COL1-3) and four noncollagenous domains (NC1-4). The human alpha1(XIII) chain contains altogether eight cysteine residues. We introduced point mutations to six of the most N-terminal cysteine residues, and we show here that the two cysteines 117 and 119 at the end of the N-terminal noncollagenous domain (NC1) are responsible for linking the three alpha1(XIII) chains together by means of interchain disulfide bonds. In addition, the intracellular and transmembrane domains have an impact on trimer formation, whereas the cysteines in the transmembrane domain and the COL1, the NC2, and the C-terminal NC4 domains do not affect trimer formation. We also suggest that the first three noncollagenous domains (NC1-3) harbor repeating heptad sequences typical of alpha-helical coiled-coils, whereas the conserved NC4 lacks a coiled-coil probability. Prevention of the coiled-coil conformation in the NC3 domain is shown here to result in labile type XIII collagen molecules. Furthermore, a new subgroup of collagenous transmembrane proteins, the Rattus norvegicus, Drosophila melanogaster, and Caenorhabditis elegans colmedins, is enlarged to contain also Homo sapiens collomin, and Pan troglodytes, Mus musculus, Tetraodon nigroviridis, and Dano rerio proteins. We suggest that there is a structurally varied group of collagenous transmembrane proteins whose biosynthesis is characterized by a coiled-coil motif following the transmembrane domain, and that these trimerization domains appear to be associated with each of the collagenous domains. In the case of type XIII collagen, the trimeric molecule has disulfide bonds at the junction of the NC1 and COL1 domains, and the type XIII collagen-like molecules (collagen types XXIII and XXV) and the colmedins are similar in that they all have a pair of cysteines in the same location. Moreover, furin cleavage at the NC1 domain can be expected in most of the proteins.     

The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 from the moderate thermophile Bacillus stearothermophilus ribosome

The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 isolated from ribosomes of the moderate thermophile Bacillus stearothermophilus are presented. This has been achieved by the sequence analysis of peptides derived by enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease, as well as by chemical cleavage with cyanogen bromide. The proteins L5 and L18 consist of 179 and 120 amino acid residues, and have Mr values of 20,163 and 13,473, respectively. A comparison of the sequences with their counterparts from the Escherichia coli ribosome reveals 59% identical residues for L5, and 53% for L18. For both proteins, the distribution of conserved regions is not random along the protein chains: some regions are highly conserved while others are not. The regions which are conserved during evolution may be important for the interaction with the 5 S rRNA molecule.     

Protein V, a novel type-II IgG receptor from Streptococcus sp.: sequence, homologies and putative Fc-binding site.

We have cloned and sequenced the Fc-receptor-encoding gene, fcrV, from a group G streptococcus. Considerable similarity was revealed between the FcRV, FcRA76 and M proteins of group A streptococci in their signal sequences and 3' termini, and between the Fc-binding regions of FcRV and FcRA76. The promoter and terminator regions showed no homology with those of the fcrA76 and M protein-encoding genes. The A1-A4 domains of FcrV (protein V) exhibit a heptapeptide repeat motif which is characteristic of alpha-helical coiled-coil proteins. The sequence, Ser-Asn-Arg-Ala-Ala, in the outer position, 'f' of each domain is highly conserved and may be involved in FcR-IgG interactions.     

An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins

Summary We have analyzed and compared the amino acid sequences of the type 4 fimbrial subunits fromPseudomonas aeruginosa, Moraxella bovis, M. nonliquefaciens, Bacteroides nodosus, Neisseria gonorrhoeae, andN. meningitidis. We propose a consensus sequence for the highly conserved aminoterminal regions of these proteins. In the variable regions, a domain corresponding to an epitope common toN. gonorrhoeae andN. meningitidis fimbriae is conserved, both in sequence and in environment, in fimbrial subunits fromB. nodosus. The subunits fromM. bovis andP. aeruginosa do not show any homologies to this sequence. In all of the subunits, the carboxy-terminal half of the molecule consists of a series of fairly hydrophobic domains. The last three domains, two of which include the cysteines of the disulfide bridge inN. gonorrhoeae, P. aeruginosa, andM. bovis, are more or less conserved in sequence in all of the proteins including that ofB. nodosus. We propose that these conserved hydrophobic regions, which have the potential to form a series of beta-sheets, form a structural framework around which more variable hydrophilic sequences determining immunological profile are arranged. The evolutionary relationships of the contemporary proteins and the distribution of type 4 fimbriae are also discussed.     

Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments.

Desmoplakins (DP) and bullous pemphigoid antigen (BPA) are major plaque components of the desmosome and hemidesmosome, respectively. These cell adhesion structures are both associated intimately with the intermediate filament (IF) network. Structural analyses of DP and BPA sequences have indicated that these molecules are likely to form extended dumbbell-shaped dimers with a central rod and globular end domains. Recent sequence data have indicated that the N-terminal domains of both DP and BPA (like their C-terminal domains) are highly related: the former contain regions of heptad repeats that are predicted to form several alpha-helical bundles. Comparisons of DP and BPA protein sequences with that of plectin (PL), a 466 kDa IF-associated protein, have also revealed large scale homology. Identities between their N-terminal domains are: DP:BPA = 35%, DP:PL = 32%, BPA:PL = 40%, suggesting that BPA is more closely related to PL than DP in this region. In the C-terminal domains, which contain a 38-residue repeating motif, however, DP and PL are closer relatives (identities: DP:BPA = 38%, BPA:PL = 40%, DP:PL = 49%). The central domains of all three proteins have extensive heptad repeat substructure, express the same periodic distribution of charged residues, and are predicted to form two-stranded alpha-helical coiled-coil ropes. These observations suggest that DP, BPA and PL belong to a new gene family encoding proteins involved in IF organization.     

Amino acid sequence of the regulatory subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase

Evidence is presented that establishes the amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle. Complementary sets of overlapping peptides were generated primarily by tryptic digestion and by chemical cleavage at methionyl residues. The analysis was augmented by chemical cleavage at a single tryptophanyl residue and at three of the four aspartyl-proline bonds. Several large fragments generated by limited proteolysis contributed to the proof of structure. The subunit is a single chain of 400 residues corresponding to a molecular weight of 45 004. An amino-terminal segment of about 100 residues is believed to include the region responsible for oligomeric association. The remainder of the molecule consists of two tandem homologous domains, each of which is thought to bind a single molecule of cAMP. Comparison of the three domains with corresponding regions of the type I isozyme, of the Escherichia coli catabolite gene activator protein, and of cGMP-dependent protein kinase indicates extensive regions of homology and as much as 50% identity with the sequence of an internal segment of the type I isozyme.     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.     

Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.     

Analysis of Heterogeneity of IgG-Binding Proteins Expressed by Group A Streptococci

Methods for analyzing the functional heterogeneity of type II IgG-binding proteins expressed by group A streptococci are described. Evidence for two major antigenic classes of type II IgG-binding proteins is presented. Heterogeneity in functional binding profiles was found to be associated with proteins belonging to either antigenic class. The antigenic class of IgG-binding protein was found to correlate with the antigenic class of M protein expressed by the same group A isolate. For certain group A isolates, the IgG-binding protein and M protein were shown to be a single molecule.     

The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion

Mitochondria are double‐membrane‐bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N‐terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane‐bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C‐terminal amphipathic tail of the Atlastin protein.