Analysis of the structure–function relationship of the S-layer protein SbsC of Bacillus stearothermophilus ATCC 12980 by producing truncated forms

Abstract

The mature surface layer (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and self-assembles into an oblique lattice type which functions as an adhesion site for a cell-associated highmolecular-mass exoamylase. To elucidate the structure-function relationship of distinct segments of SbsC, three N-and seven C-terminal truncations were produced in a heterologous expression system, isolated, purified and their properties compared with those of the recombinant mature S-layer protein rSbsC 31-1099 . With the various truncated forms it could be demonstrated that the N-terminal part (aa 31-257) is responsible for anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, but this positively charged segment is not required for the self-assembly of SbsC, nor for generating the oblique lattice structure. If present, the N-terminal part leads to the formation of in vitro double-layer self-assembly products. Affinity studies further showed that the N-terminal part includes an exoamylase-binding site. Interestingly, the N-terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) that were also identified on the amylase-binding protein AbpA of Streptococcus gordonii. In contrast to the self-assembling N-terminal truncation rSbsC 258-1099 , two further N-terminal truncations (rSbsC 343-1099 , rSbsC 447-1099 ) and three C-terminal truncations (rSbsC 31-713 , rSbsC 31-844 , rSbsC 31-860 ) had lost the ability to self-assemble and stayed in the water-soluble state. Studies with the self-assembling C-terminal truncations rSbsC 31-880 , rSbsC 31-900 and rSbsC 31-920 revealed that the C-terminal 219 aa can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure.

Phylogenetic Analysis and Heterologous Expression of Surface Layer Protein SlpC ofBacillus sphaericusC3-41

“…This finding is in accordance with previous results that S-layer proteins formed sheet-like or fingerprint-shape structures in cytoplasm of the host cells. 24) No self-assembly product was observed in E. coli BL21 containing pET28a ( Fig. 4c and d), but negative staining study showed that the purified SlpC and ÁSlpC fusion protein had no sheet-like crystal lattice structure.…”

The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together

“…All other investigated strains, such as G. stearothermophilus PV72/p6 (SbsA; Kuen et al, 1994;X71092) For the S-layer protein SbsC it was shown experimentally that a positively charged N-terminal fragment (amino acids 31-257) is responsible for anchoring the S-layer subunits. Using several truncated forms of SbsC it was demonstrated that this region is not required either for self-assembly or for generating the oblique lattice structure (Jarosch et al, 2001). Interestingly, none of the SCWPs of the organisms that possess S-layer proteins without SLH domains are modified with pyruvyl groups and some of them have a net-neutral charge (see Fig.…”

Genetic analysis of Bacillus anthracis Sap S-layer protein crystallization domain

“…Electron microscopy and image analysis procedures have shown that all S-layer proteins possess two morphological domains (Baumeister et al, 1989;Engelhardt & Peters, 1998). Biochemical, biophysical and genetic approaches have shown that one of these domains is the cellwall-anchoring domain (Lupas et al, 1994;Mesnage et al, 1999;Jarosch et al, 2001;Smit et al, 2001;Rünzler et al, 2004). Gram-positive bacteria contain various anchoring domains that interact with different secondary cell wall polymers.…”

“…The other domain appears to be a crystallization domain (Bingle et al, 1987;Mesnage et al, 1997Mesnage et al, , 1999Howorka et al, 2000;Jarosch et al, 2001;Smit et al, 2002;Mader et al, 2004). Recent studies have focused on the structurefunction relationships of this domain.…”

“…Recent studies have focused on the structurefunction relationships of this domain. Mutagenesis approaches, be they point mutations or deletions, have identified regions within the crystallization domain that are critical for array assembly (Howorka et al, 2000;Sillanpää et al, 2000;Jarosch et al, 2001;Smit et al, 2002). However, these analyses have not provided an overall picture of how an S-layer protein self-assembles to generate an ordered array.…”