title: The surface location of individual residues in a bacterial S-layer protein.
abstract: Bacterial surface layer (S-layer) proteins self-assemble into large two-dimensional crystalline lattices that form the outermost cell-wall component of all archaea and many eubacteria. Despite being a large class of self-assembling proteins, little is known about their molecular architecture. We investigated the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 to identify residues located at the subunit-subunit interface and to determine the S-layer's topology. Twenty-three single cysteine mutants, which were previously mapped to the surface of the SbsB monomer, were subjected to a cross-linking screen using the photoactivatable, sulfhydryl-reactive reagent N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide. Gel electrophoretic analysis on the formation of cross-linked dimers indicated that 8 out of the 23 residues were located at the interface. In combination with surface accessibility data for the assembled protein, 10 residues were assigned to positions at the inner, cell-wall-facing lattice surface, while 5 residues were mapped to the outer, ambient-exposed lattice surface. In addition, the cross-linking screen identified six positions of intramolecular cross-linking within the assembled protein but not in the monomeric S-layer protein. Most likely, these intramolecular cross-links result from conformational changes upon self-assembly. The results are an important step toward the further structural elucidation of the S-layer protein via, for example, X-ray crystallography and cryo-electron microscopy. Our approach of identifying the surface location of residues is relevant to other planar supramolecular protein assemblies.
authors.affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London WC1H 0AJ, England, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Kinns
authors.forename: Stefan
authors.initials: S
authors.lastname: Howorka
mesh_terms: D001406:Bacillaceae / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004164:Diphosphonates / Q000737:chemistry; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004164:Diphosphonates; D008565:Membrane Proteins; D011994:Recombinant Proteins; C031876:azacycloheptane-2,2-diphosphonate; D003545:Cysteine