Identification of two binding domains, one for peptidoglycan and another for a secondary cell wall polymer, on the N-terminal part of the S-layer protein SbsB from Bacillus stearothermophilus PV72/p2 - PubMed (original) (raw)

Identification of two binding domains, one for peptidoglycan and another for a secondary cell wall polymer, on the N-terminal part of the S-layer protein SbsB from Bacillus stearothermophilus PV72/p2

M Sára et al. J Bacteriol. 1998 Dec.

Abstract

First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

SDS-polyacrylamide gel electrophoresis pattern of cleavage fragments formed by limited proteolysis of the S-layer protein from B. stearothermophilus PV72/p2 with endoproteinase Glu-C in 0.1% SDS (lane A) and 2 M GHCl (lanes B and C) in 50 mM Tris-HCl buffer (pH 7.2) in the absence (lane B) and the presence (lane C) of the SCWP. Conditions for proteolytic cleavage were as follows. One milligram of S-layer protein was dissolved in 1 ml of 0.1% SDS or 2 M GHCl, 40 μg of endoproteinase Glu-C was added, and samples were incubated for 1 h at 37°C. The concentration of the SCWP was 250 μg per 1 mg of S-layer protein. Proteolytic cleavage fragments subjected to N-terminal sequencing are indicated by arrows.

FIG. 2

FIG. 2

Affinity studies performed with proteolytic cleavage fragments prepared with endoproteinase Glu-C in 0.1% SDS in 50 mM Tris-HCl buffer (pH 7.2) and native peptidoglycan-containing sacculi. Shown are proteolytic cleavage fragments before incubation with native peptidoglycan-containing sacculi (lane A), remaining in the clear supernatant recognizing as a binding site native peptidoglycan-containing sacculi (lane B), and (lane C). Lane B, two minor cleavage fragments with apparent molecular weights of 57,000 and 52,000 (V-P-V-Q-V and T-K-P-V-D-F) starting with either amino acid 355 or amino acid 409 of the mature S-layer protein. For the affinity studies, 1 mg of peptidoglycan-containing sacculi per mg of S-layer protein was added. Lane D, molecular weight standard (molecular weights given are multipliers of 1,000). Proteolytic cleavage fragments subjected to N-terminal sequencing are indicated by arrows.

FIG. 3

FIG. 3

Affinity studies with proteolytic cleavage fragments prepared in 2 M GHCl in 50 mM Tris-HCl buffer (pH 7.2) and native (lanes a through c and lanes g through i) or HF-extracted (lanes d through f) peptidoglycan-containing sacculi. In lanes g through i, the polymer binding domain on the S-layer protein was blocked by addition of SCWP (250 μg/mg of S-layer protein). Shown are proteolytic cleavage fragments before incubation with peptidoglycan-containing sacculi (lanes a, d, and g), remaining in the clear supernatant (lanes b, e, and h), and recognizing native (lanes c and i) and HF-extracted (lane f) peptidoglycan-containing sacculi as a binding site. Molecular weights given are multipliers of 1,000.

FIG. 4

FIG. 4

Schematic drawing of the S-layer protein from B. stearothermophilus PV72/p2 showing the location of the peptidoglycan and SCWP binding domain and the different endoproteinase Glu-C cleavage sites (positions of glutamic acid residues are given). The mature S-layer protein consists of 889 amino acids. ±, with or without SCWP.

Similar articles

Cited by

References

    1. Altschul S F, Madden T L, Schäffer A A, Zhang J, Zhang Z, Miller W, Lipman D J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. - PMC - PubMed
    1. Archibald A R, Hancock I C, Harwood C R. Cell wall structure, synthesis, and turnover. In: Sonenshein A, Hoch J A, Losick R, editors. Bacillus subtilis and other gram-positive bacteria. Washington, D.C: American Society for Microbiology; 1993. pp. 381–410.
    1. Beveridge T J. Bacterial S-layers. Curr Opin Struct Biol. 1994;4:204–212.
    1. Beveridge T J, Pouwels P H, Sára M, Kotiranta A, Lounatmaa K, Kari K, Kerosuo E, Happasalo M, Egelseer E M, Schocher I, Sleytr U B, Morelli L, Callegari M-L, Nomellini J F, Bingle W H, Smit J, Leibovitz E, Lemaire M, Miras I, Salamitou S, Beguin P, Ohayon H, Gounon P, Matuschek M, Sahm K, Bahl H, Grogono-Thomas R, Dworkin J, Blaser M J, Woodland R M, Newell D G, Kessel M, Koval S F. Functions of S-layers. FEMS Microbiol Rev. 1997;20:99–149. - PubMed
    1. Bowditch R D, Baumann P, Yousten A A. Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene. J Bacteriol. 1989;171:4178–4188. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources