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 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.
Jarosch
Marina
M
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
Egelseer
Eva M
EM
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
Huber
Carina
C
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
Moll
Dieter
D
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
Mattanovich
Diethard
D
Institute of Applied Microbiology, University of Agricultural Sciences, 1190 Vienna, Austria2.
Sleytr
Uwe B
UB
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
Sára
Margit
M
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1.
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; C055764:S-layer proteins