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.
Figures
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
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
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
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.
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