Towards the structure of the C-terminal part of the S-layer protein SbsC

Abstract

The S-layer protein SbsC from Geobacillus stearothermophilus ATCC 12980 is the most prevalent single protein produced by the bacterium and covers the complete bacterial surface in the form of a two-dimensional crystalline monolayer. In order to elucidate the structural features of the assembly domains, several N-terminally truncated fragments of SbsC have been crystallized. Crystals obtained from recombinant fragments showed anisotropic diffraction to a maximum of 3.5 Å resolution using synchrotron radiation. The best diffracting crystals were obtained from rSbsC , an unintentional in situ proteolytic degradation product of rSbsC . Crystals were obtained in two different space groups, P2 1 and P4 1 2 1 2, and diffracted to 2.6 and 3 Å resolution, respectively. Native and heavy-atom derivative data have been collected. The structure of the C-terminal part will yield atomic resolution information for the domains that are crucial for the assembly of the two-dimensional lattice.

Crystallization of domains involved in self-assembly of the S-layer protein SbsC

“…The structures of two SbsC C-terminal truncation constructs and the successful crystallization of the N-terminally truncated construct rSbsC (755-1099) have been reported previously (Pavkov et al, 2008;Kroutil et al, 2009). The structure of rSbsC , containing the first three N-terminal domains, has been determined to 2.4 Å resolution P2 1 P4 1 2 1 2 P4 1 2 1 2 P2 1 Resolution limits (Å ) 19.9-3.…”

“…The truncation constructs rSbsC (31-761) , rSbsC (31-754) , rSbsC (31-790) , rSbsC (447-754) , rSbsC (443-650) and rSbsC (541-759) were cloned and expressed according to published protocols (Jarosch et al, 2001;Kroutil et al, 2009). In brief, the gene sequences encoding the truncations were PCR-amplified using the respective oligonucleotide primers (given in Table 1), which introduced the restriction sites NcoI (including an ATG start codon) and XhoI at the 5 0 and 3 0 ends, respectively.…”

“…The property of S-layer proteins to self-assemble into twodimensional crystals makes them very demanding candidates for structural studies. The crystallization and/or X-ray structures of several bacterial and archaeal truncated and soluble S-layer constructs that have been reported to date are as follows: N-terminal and C-terminal parts of SbsC from G. stearothermophilus ATTC 12980 (Kroutil et al, 2009;Pavkov et al, 2003Pavkov et al, , 2008, a truncated derivative of the low-molecular-weight (LMW) S-layer protein from Clostridium difficile (Fagan et al, 2009), the cell-wall-binding domain of the Sap protein from Bacillus anthracis comprised of three S-layer homology (SLH) motifs (Kern et al, 2011) and polypeptide chain constructs from the archaeal surface-layer proteins from Staphylothermus marinus (Stetefeld et al, 2000), Methanosarcina mazei (Jing et al, 2002) and M. acetivorans (Arbing et al, 2012). Recently, the X-ray structure of the S-layer protein SbsB from G. stearothermophilus PV72/p2 lacking the cell-wall-binding domain was reported (Baranova et al, 2012).…”