Anchor structure of staphylococcal surface proteins. A branched peptide that links the carboxyl terminus of proteins to the cell wall

Surface proteins of Staphylococcus aureus are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal. Previous work demonstrated that the sorting signal is cleaved at the conserved LPXTG motif and that the carboxyl of threonine (T) is linked to the staphylococcal cell wall. By employing different cell wall lytic enzymes, surface proteins were released from the staphylococcal peptidoglycan and their COOH-terminal anchor structure was revealed by a combination of mass spectrometry and chemical analysis. The results demonstrate that surface proteins are linked to a branched peptide (NH2-Ala-gamma-Gln-Lys-(NH2-Gly5)-Ala-COOH) by an amide bond between the carboxyl of threonine and the amino of the pentaglycine cross-bridge that is attached to the epsilon-amino of lysyl. This branched anchor peptide is amide-linked to the carboxyl of N-acetylmuramic acid, thereby tethering the COOH-terminal end of surface proteins to the staphylococcal peptidoglycan.     

Anchor structure of staphylococcal surface proteins. III. Role of the FemA, FemB, and FemX factors in anchoring surface proteins to the bacterial cell wall

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the pentaglycyl cross-bridge in the staphylococcal peptidoglycan. Here, we asked whether altered peptidoglycan cross-bridges interfere with the sorting reaction and investigated surface protein anchoring in staphylococcal fem mutants. S. aureus strains carrying mutations in the femA, femB, femAB, or the femAX genes synthesize altered cross-bridges, and each of these strains displayed decreased sorting activity. Characterization of cell wall anchor structures purified from the fem mutants revealed that surface proteins were linked to cross-bridges containing one, three, or five glycyl residues, but not to the epsilon-amino of lysyl in muropeptides without glycine. When tested in a femAB strain synthesizing cross-bridges with mono-, tri-, and pentaglycyl as well as tetraglycyl-monoseryl, surface proteins were found anchored mostly to the five-residue cross-bridges (pentaglycyl or tetraglycyl-monoseryl). Thus, although wild-type peptidoglycan appears to be the preferred substrate for the sorting reaction, altered cell wall cross-bridges can be linked to the COOH-terminal end of surface proteins.     

Probing protein structure by solvent perturbation of NMR spectra. II. Determination of surface and buried residues in homologous proteins

The experimental assignment of most residues in a protein to the surface or interior is in principle possible without prior solution of a complete three-dimensional structure. The method described is based on nmr measurements that determine the amino acid composition of the surface of a protein [A. Petros, L. Mueller, and K.D. Kopple (1990) Biochemistry, Vol. 29, pp. 10041-10048; G. Esposito, A. M. Lesk, H. Molinari, A. Motta, N. Niccolai, and A. Pastore (1992) Journal of Molecular Biology, Vol. 224, pp. 659-670]. If these measurements are carried out on several homologous proteins of known sequence, it is possible to combine the results to determine, in most cases, which positions in the sequence contain exposed residues.     

Expression and native structure of cytosolic class II small heat-shock proteins

Higher plants synthesize small heat-shock proteins (smHSPs) from five related gene families. The class I and II families encode cytosolic smHSPs. We characterized the class II smHSPs of pea (Pisum sativum) and compared them with class I smHSPs. Antibodies against recombinant HSP17.7, a class II smHSP, recognized four heat-inducible 17- to 18-kD polypeptides and did not cross-react with class I smHSPs. On sucrose gradients the class II smHSPs sedimented primarily at 8 Svedberg units, indicating that they are components of large complexes similar in size to class I smHSP complexes. However, the class I and II complexes were readily distinguishable by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Nondenaturing immune precipitations using anti-HSP17.7 or anti-HSP18.1 (a class I smHSP) antiserum provide further evidence that the class I and II smHSPs exist in different complexes, composed primarily of smHSPs. Recombinant HSP17.7 and HSP18.1 formed complexes of sizes similar to those formed in vivo. When these two smHSPs were mixed, denatured with urea, and then dialyzed, the distinct class I and II complexes again formed, each containing only HSP18.1 or HSP17.7. Thus, cytosolic smHSPs from two related gene families expressed simultaneously form distinct complexes in vivo, suggesting that they have subtly different functions.     

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.     

The structure and characterization of the surface protein layer of a Comamonas-like organism

The regular surface layer of a strain of a Comamonas-like organism was examined by electron microscopy. The surface layer protein was easily extracted from the cell surface by a 2.5 M solution of lithium chloride. The protein subunit has a molecular size of 32,000 daltons, but usually forms a large aggregate of more than 1,200,000 daltons. In the extract it formed a regular array of p4 symmetry and was observed to be intimately associated with fragments of lipopolysaccharide. The size of a subunit determined by the negative staining method and the image processing method measured 5.2 x 6.4 nm (width and length), was arranged in a cobblestone-like pattern, and was located in a lattice space measuring 13.0 nm square.     

Discrimination of structure: II. Feature binding.

In 3 experiments, pigeons acquired a discrimination between patterns comprising the same features. Thus vertical green bars beside horizontal red bars might have signaled food, and horizontal green bars beside vertical red bars might have signaled no food. The solution of this discrimination can be explained by assuming each pattern is represented either by a template or by structural features that are sensitive to combinations of color and line orientation. The 1st explanation predicts subjects should react to a training pattern rotated 90° in the same way as the pattern on which it is based. The 2nd explanation predicts these patterns should be treated as if they signal opposing outcomes. The experiments confirmed the 2nd of these predictions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure

A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding alpha subunit of the inhibitory glycine receptor forms anion-selective channels in phospholipid bilayers. The most frequent events show single-channel conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau o, in the millisecond time range. These properties match those of authentic glycine receptors studied in inside-out patches of cultured rat spinal cord neurons, namely gamma = 27 pS and gamma = 45 pS, and tau o in the millisecond time range. The channel activity of M2GlyR is sequence-specific: 1) a synthetic peptide with the sequence of putative transmembrane segment M1 (M1GlyR), not considered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays distinct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2GlyR) forms cation-selective channels. Further, a four-helix bundle protein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gamma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, but not by an analog of the local anesthetic lidocaine (QX-222), a cation channel blocker. Observed single-channel properties suggest that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M2 could confer anion specificity to the receptor channel.     

SMC proteins and chromosome structure

The structure of chromosomes is largely determined by chromosome-associated proteins. Members of the SMC (structural maintenance of chromosomes) family play an important role in both prokaryotic and eukaryotic chromosome structure and dynamics. SMC proteins are involved in chromosome condensation, sister-chromatid cohesion, sex-chromosome dosage compensation, genetic recombination and DNA repair. There have been major advances recently in understanding the function of SMC proteins--including the identification of biochemical activities of SMC-containing protein complexes and the realization that individual SMC proteins might link seemingly unrelated aspects of chromosomal metabolism.     

Sequence diversity, predicted two-dimensional protein structure, and epitope mapping of neisserial Opa proteins

The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane beta-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.     

Modeling unfolded states of proteins and peptides. II. Backbone solvent accessibility

Buried surface area is often used as a measure of the contribution to protein folding from the hydrophobic effect. Quantitatively, the surface buried upon folding is reckoned as the difference in area between the native and unfolded states. This calculation is well defined for a known structure but model-dependent for the unfolded state. In a previous paper [Creamer, T. P., Srinivasan, R., & Rose, G. D. (1995) Biochemistry 34, 16245-16250], we developed two models that bracket the surface area of the unfolded state between limiting extremes. Using these extrema, it was shown that earlier models, such as an extended tripeptide, overestimate the surface area of side chains in the unfolded state. In this sequel to our previous paper, we focus on backbone surface in the unfolded state, again adopting the strategy of trapping the area between limiting extrema. A principal conclusion of this present study is that most backbone surface in proteins is buried within local structure.     

Cell wall formation in zoospores of Allomyces arbuscula. II. Development of surface structure of encysted haploid zoospores, rhizoids, and hyphae

Development of haploid meiospores of Allomyces arbuscula into germling cells with rhizoids and hyphae was followed during incubation in complete growth medium. The surface structure of encysted meiospores, rhizoids and hyphae before and after extraction of amorphous materials with ethanolic KOH was studied by means of carbon-platinum replicas. After 2--3 min incubation in complete medium 10% of the meiospores were surrounded by a cell wall containing microfibrils embedded in a matrix. Structure of cell walls of encysted meiospores, rhizoids, and hyphae differ from one another by the location of amorphous materials and by the arrangement of chitin microfibrils.     

Computer-aided studies on the spatial structure of the small hepatitis B surface protein

Progressive Multifocal Leukoencephalopathy (PML) is a subacute demyelinating opportunistic infection of the central nervous system which frequently occurs in AIDS. CT and MR, together with clinical and virological findings, can suggest a correct diagnosis in most cases, thus avoiding stereotactic biopsy which is too invasive considering the lack of therapy and the poor prognosis of this disease. In this study we reviewed the CT and MR findings of 16 proved AIDS-related PML cases. PML lesions appeared as hypodense on CT, hypointense on T1w, hyperintense on PDw and T2w MR images. CT was less sensitive than T2w MR images and underestimated the number of lesions and/or disease extent. On the basis of our findings during the progression of the lesions we observed two different patterns of PML presentation and evolution i.e., "single" and "multifocal". Single lesions generally involve subcortical white matter (arcuate fibers) of parietal lobe and spread to the contralateral hemisphere across the corpus callosum; multiple "patchy" lesions can be localized variably in the cerebral hemispheres and also in the brainstem and cerebellar hemispheres.     

Detection of muramidase (lysozyme)-secreting leucocytes at the single-cell level by a protein A plaque assay

Using a modification of the protein A plaque assay, muramidase (lysozyme)-producing leucocytes were detected as plaque-forming cells. In the presence of anti-muramidase Ig and complement the secreted lysozyme resulted in lysis of protein-A-coated target erythrocytes. By the use of a monolayer technique individual plaque-forming cells could be identified by staining procedures. Granulocytes as well as monocytes were found to produce muramidase and thus to form plaques. This method could serve as a useful tool when studying lysozyme secretion. Furthermore, by the use of appropriate antisera, this method could be employed for the study of any cell type (any secretion), provided enough molecules are being secreted.     

The solution structure of the bovine leukaemia virus matrix protein and similarity with lentiviral matrix proteins

In the mature virion, retroviral matrix proteins are found in association with the inner face of the viral membrane. They play a critical role in determining the morphogenesis of virus assembly. We have determined the three-dimensional solution structure of the bovine leukaemia virus (BLV) matrix protein by heteronuclear nuclear magnetic resonance. The protein contains four principal helices that are joined by short, partially structured loops. Despite no sequence similarity with the lentiviruses, the structure shows an intriguing homology with the equivalent protein from the human and simian immunodeficiency viruses. A root-mean-square deviation of 3.78 angstrom is observed over the backbone atoms of 36 equivalent helical positions. The similarity implies a possible common assembly unit for the matrix proteins of type C retroviruses.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.