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

The mature surface layer (S-layer) protein SbsC of Bacillus stearothermophi lus ATCC 12980 comprises amino acids 31-1099 and self-assembles into an obl ique lattice type which functions as an adhesion site for a cell-associated high-molecular-mass exoamylase. To elucidate the structure-function relati onship of distinct segments of SbsC, three N- and seven C-terminal truncati ons were produced in a heterologous expression system, isolated, purified a nd their properties compared with those of the recombinant mature S-layer p rotein rSbsC(31-1099). With the various truncated forms it could be demonst rated 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 stru cture. If present, the N-terminal part leads to the formation of in vitro d ouble-layer self-assembly products. Affinity studies further showed that th e N-terminal part includes an exoamylase-binding site. Interestingly, the N -terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) tha t were also identified on the amylase-binding protein AbpA of Streptococcus gordonii, In contrast to the self-assembling N-terminal truncation rSbsC(2 58-1099), two further N-terminal truncations (rSbsC(343-1099), rSbsC(447-10 99)) 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-solu ble 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 t he C-terminal 179 aa are not required for the formation of the oblique latt ice structure.