Jarosch Marina, Egelseer Eva M, Huber Carina, Moll Dieter, Mattanovich Diethard, Sleytr Uwe B, Sára Margit
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
Institute of Applied Microbiology, University of Agricultural Sciences, 1190 Vienna, Austria2.
Microbiology (Reading). 2001 May;147(Pt 5):1353-1363. doi: 10.1099/00221287-147-5-1353.
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 high-molecular-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.
嗜热脂肪芽孢杆菌ATCC 12980的成熟表面层（S层）蛋白SbsC由氨基酸31 - 1099组成，能自组装成一种斜晶格类型，作为细胞相关高分子量外切淀粉酶的粘附位点。为阐明SbsC不同区段的结构 - 功能关系，在异源表达系统中产生了三个N端和七个C端截短体，进行分离、纯化，并将其性质与重组成熟S层蛋白rSbsC(31 - 1099)的性质进行比较。通过各种截短形式可以证明，N端部分（氨基酸31 - 257）负责通过一种独特类型的次生细胞壁聚合物将S层亚基锚定到刚性细胞壁层，但这个带正电荷的区段对于SbsC的自组装以及形成斜晶格结构并非必需。如果存在，N端部分会导致体外双层自组装产物的形成。亲和研究进一步表明，N端部分包含一个外切淀粉酶结合位点。有趣的是，N端部分带有两个6个和7个氨基酸的序列（AKAALD和KAAYEAA），这两个序列也在戈登链球菌的淀粉酶结合蛋白AbpA上被鉴定出来。与能自组装的N端截短体rSbsC(258 - 1099)不同，另外两个N端截短体（rSbsC(343 - 1099)，rSbsC(447 - 1099)）和三个C端截短体（rSbsC(31 - 713)，rSbsC(31 - 844)，rSbsC(31 - 860)）失去了自组装能力，处于水溶性状态。对能自组装的C端截短体rSbsC(31 - 880)、rSbsC(31 - 900)和rSbsC(31 - 920)的研究表明，C端的219个氨基酸可以被删除而不干扰自组装过程，而C端的179个氨基酸对于斜晶格结构的形成并非必需。
