Exploring structural homology of proteins.

A method for systematically comparing the folding of the three-dimensional structures of proteins has been developed. A search function, plotted in terms of three Eulerian angles, represents the number of sequentially equivalenced amino acids. For each orientation one protein structure is rotated about its center of mass with respect to the other and probabilities are calculated which estimate the degree of structural parallelism. The structurally equivalent residues with highest probabilities are then selected for the best common topology. It was observed that, when structures containing about 150 residues were compared, the random background had a mean value of around 14 residues and the standard deviation was approximately nine residues. The method has been shown to be successful in determining the similarity of the NAD binding domains of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, and in comparing the heme binding fold of cytochrome b₅ with the globins.Application of the method to compare hen egg white lysozyme and T4 phage lysozyme led to a single significant peak of 62 residues. The structural homology indicated by this peak showed that the substrate, as bound to hen egg white lysozyme, has a corresponding binding site in the large cleft of the phage lysozyme. The predicted binding site of N-acetyl glucosamine at position C compares well with an N-acetyl glucosamine center observed to bind to crystalline phage lysozyme (B. W. Matthews, personal communication).Some results for the comparison of the two Fe-S cage binding domains of ferredoxin are also presented.     

Sequence homology between two immunodiagnostic fusion proteins from Echinococcus multilocularis.

Serological diagnosis of alveolar hydatid disease using crude parasite antigen is problematical with the result that several groups have addressed the question of specific serodiagnosis using defined native or recombinant antigens. Sequence data are available for two lambda gt 11 cDNA clones, designated EM4 and pEM10, which express E. multilocularis species-specific proteins in immunodiagnostic assays using human sera. Here, we have drawn attention to the extensive similarities between these antigens and have compared their characteristics since both may prove valuable in the future diagnosis of alveolar hydatidosis.     

Sequence homology between Moloney murine sarcoma virus and Moloney leukemia virus RNA.

The Moloney murine sarcoma-leukemia virus [M-MSV (MuLV)], propagated at high multiplicity of infection (MOI), was demonstrated previously to contain a native genome mass of 4 X 10(6) daltons as contrasted to a mass of 7 X 10(6) daltons for Moloney murine leukemia virus (M-MuLV). The 4 X 10(6)-dalton classof RNA from M-MSV (MuLV) was examined for base sequence homology with DNA complementary to the 7 X 10(6)-dalton M-MuLV RNA genome. Approximately 86% of the M-MSV (MuLV) was protected from RNase digestion by hybridization, whereas 95% of M-MuLV was protected under identical conditions. These results indicate that the small RNA class of high-MOI M-MSV (MuLV) contains little (perhaps 10%) genetic information not present in M-MuLV. Virtually all of the 1.8 X 10(6)-dalton subunits of M-MSV (MuLV) RNA contained regions of poly(A) since 94% of the RNA bound to oligo(dT) cellulose in 0.5 M KCl. This suggests that the formation of the 1.8 X 10(6)-dalton subunits occurs before their packaging into virions and does not result from hydrolysis of intact 3.5 X 10(6)-dalton subunits by a virion-associated nuclease.     

Characterization of the deoxyribonucleic acid polymerase associated with Kilham rat virus.

Purified preparations of the parvovirus, Kilham rat virus, have associated with them a protein with DNA polymerase activity. The enzyme has been separated from the other two or three viral proteins and purified 63-fold. The viral associated enzyme was found in a single peak of DNA polymerase activity after chromatography on DEAE-cellulose, DNA-cellulose, and phosphocellulose columns. It shares some properties in common with the host cellular DNA polymerases, described in the preceding paper (Salzman, L.A., and McKerlie, L. (1975) J. Biol. Chem. 250, 5589-5595), but also has some important distinguishing characteristics. The Kilham rat virus-associated DNA polymerase has increased enzyme activity in the presence of 0.02 M KCl and has a strong preference for a synthetic DNA polymer containing deoxyadenylate and deoxythymidylate. The enzyme has a molecular weight of approximately 75,000 plus or minus 3,000 and appears to contain endonuclease activity.     

Sequence homology and structural analysis of the clostridial neurotoxins.

The clostridial neurotoxins (CNTs), comprised of tetanus neurotoxin (TeNT) and the seven serotypes of botulinum neurotoxin (BoNT A-G), specifically bind to neuronal cells and disrupt neurotransmitter release by cleaving proteins involved in synaptic vesicle membrane fusion. In this study, multiple CNT sequences were analyzed within the context of the 1277 residue BoNT/A crystal structure to gain insight into the events of binding, pore formation, translocation, and catalysis that are required for toxicity. A comparison of the TeNT-binding domain structure to that of BoNT/A reveals striking differences in their surface properties. Further, the solvent accessibility of a key tryptophan in the C terminus of the BoNT/A-binding domain refines the location of the ganglioside-binding site. Data collected from a single frozen crystal of BoNT/A are included in this study, revealing slight differences in the binding domain orientation as well as density for a previously unobserved translocation domain loop. This loop and the conservation of charged residues with structural proximity to putative pore-forming sequences lend insight into the CNT mechanism of pore formation and translocation. The sequence analysis of the catalytic domain revealed an area near the active-site likely to account for specificity differences between the CNTs. It revealed also a tertiary structure, highly conserved in primary sequence, which seems critical to catalysis but is 30 A from the active-site zinc ion. This observation, along with an analysis of the 54 residue "belt" from the translocation domain are discussed with respect to the mechanism of catalysis.     

Isolation and structural characterization of monomeric and dimeric forms of replicative intermediates of Kilham rat virus DNA.

Two virus-specific species of newly synthesized DNA were isolated from rat fibroblast cell cultures infected with the Kilham rat virus (RV). These two DNA species were purified; their behavior on hydroxyapatite chromatography and their sedimentation coefficients in sucrose gradients were determined. One of the two species corresponds to the linear double-stranded form of the RV DNA, and the other corresponds to the dimeric duplex form. After denaturation, a fraction of both species showed an intramolecular renaturation; these molecules are composed of viral strand covalently linked to complementary strand. Models for the structure of both species are posposed. Both species may be considered as double-strand replicative intermediates of the single-stranded RV DNA.     

Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology.

The complete nucleotide sequence of the NS mRNA of vesicular stomatitis virus (New Jersey serotype) was established from two cDNA clones spanning the entire coding region of the mRNA. The gene is 856 nucleotides long and can code for a polypeptide of 274 amino acids. Comparison with the nucleotide sequence of the NS gene of the Indiana serotype revealed only 41% sequence homology. The deduced amino acid sequences of the NS proteins were only 32% homologous, with no identical stretches of more than five amino acids. However, at the C-terminal domain there was a conserved region of 21 amino acids with greater than 90% homology. Surprisingly, relative hydropathicity plots also demonstrated the presence of a large number of hydrophilic amino acids sequestered similarly over the N-terminal half of the protein. In addition, the total number of serine and threonine residues, presumptive phosphorylation sites, was similar and included seven serine and three threonine residues located at identical positions. It appears that during divergent evolution of these two vesicular stomatitis virus serotypes from a common ancestor, considerable mutation occurred in the main body of the gene but the overall structure of the protein was retained. The function of the NS protein in relation to the evolution of the two viruses is discussed.     

Sequence and structural homology among membrane-associated domains of CFTR and certain transporter proteins

A sequence comparison of the two membrane-associated (MA) domains of the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance transporter (MDR), and -factor pheromone export system (STE6) proteins, each of which are believed to contain a total of 12 transmembrane (TM) segments, reveals significant amino acid homology and length conservation in the loop regions that connect individual TM sequences. Similar structural homology is observed between these proteins, hemolysin B (HLYB) and the major histocompatibility-linked peptide transporter, HAM1, the latter two which contain a single MA domain composed of six TM segments. In addition, there are specific sequences that are conserved within the TM segments of the five different membrane proteins. This observation suggests that the folding topologies of the MA domains of MDR, STE6, and CFTR in the plasma membrane are likely to be very similar. The sequence analysis also reveals that there are three characteristic motifs (a pair of aromatic residues, LTLXXXXXXP and GXXL) that are conserved in MDR, STE6, HLYB, HAM1, but not in CFTR. We propose that although CFTR may be evolutionarily related to these other membrane proteins, it belongs to a separate subclass.     

Sequence analysis of major structural proteins of newly isolated mouse hepatitis virus.

We have isolated the virus from a fecal pellet in the colon of a BALB/c mouse with X-linked immunodeficiency (xid) housed in a room in which there has recently been an epidemic due to mouse hepatitis virus (MHV) and designated it as the MHV-TY strain. Sequence analysis of the MHV-TY strain was performed on major structural, spike (S), membrane (M) and nucleocapsid (N), proteins directly from PCR products. The comparison of nucleotide sequences of MHV-TY with other strains investigated so far revealed that all three structural proteins of the TY strain had some unique amino acid sequences among MHV strains which can be used as markers of this strain.     

Interspecies homology of RNA tumor virus proteins.

We report the application of a highly sensitive column chromatographic technique to the comparison of tryptic peptide maps of some RNA tumor virus proteins. By combining microbore ion-exchange chromatography with a sensitive fluorescent assay using o-phthalaldehyde, we obtained high-resolution peptide maps starting with only microgram amounts of protein. Our discovery of coincident peptides from the 15,000 and 30,000 molecular weight proteins from murine and feline leukemia viruses supports serological evidence for interspecies antigenic determinants; coincident peptides were also found for the 10,000 molecular weight proteins from these viruses, although immunochemical data did not reveal interspecies determinants. The relatively large number of coeluting peptides found in the 15,000 and 10,000 molecular weight proteins is strong evidence for the existence of homology.     

Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.

Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.     

Junin virus structural proteins.

Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined.     

Sequence homology between bovine and human adenoviruses.

Cross-hybridization has been detected between corresponding regions of the genomes of bovine adenovirus type 3 and human adenovirus type 2. The most conserved region on the viral genomes encodes the hexon polypeptide. The nucleotide sequence of this region in bovine adenovirus type 3 has been determined. Comparison of the predicted amino acid sequences of the bovine adenovirus type 3 and human adenovirus type 2 hexon polypeptides reveals three regions of nonhomology.     

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.     

Sequence homology among the coat proteins of gemini viruses

The coat proteins of the gemini viruses - African cassava latent virus, tomato golden mosaic virus and maize streak virus - are shown to have reasonable to good amino acid sequence homology. It is suggested that the maize streak virus genome is ancestral and the bipartite genomes of the other viruses evolved from it.     

Sequence homology between mitochondrial DNAs of different eukaryotes.

The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization. mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA. Single-stranded and duples DNA were separated by hydroxylapatite chromatography. The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns. Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken. Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess. Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex. There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to [3H]mtDNA was varied between 1250 and 7500. Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation. Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation. Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.     

Secondary infection of rat lungs with Pasteurella pneumotropica after Kilham rat virus infection

Lung congestion was observed after an outbreak of Kilham rat virus infection (KRV) in a rat colony, previously free of all rat viruses. A high proportion of congested lungs contained Pasteurella pneumotropica suggesting that KRV might have caused primary damage to the alveoli (hitherto not recorded) which allowed the secondary bacterial colonization. Experimental infection of rats with KRV caused acute damage to the lung alveoli. Since KRV infection is very common in animal facilities it could therefore be a significant agent in the development of respiratory disease.