Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

Abstract

Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many Eubacteria and most Archaea species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium Lactobacillus brevis ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG) 12 (molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW ‫؍‬ 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of Lactobacillus S-layers.Bacterial surface layers (S-layers) are cell envelope structures ubiquitously found in gram-positive and gram-negative bacteria as well as in Archaea. S-layers are composed of identical (glyco)protein subunits with a molecular mass in the range of 40 to 200 kDa. The proteins self-assemble into two-dimensional crystalline structures with oblique (p1, p2), square (p4), or hexagonal (p3, p6) symmetry, covering the entire cell surface. The subunits are held together and attached to the underlying cell wall by noncovalent interactions and they have an intrinsic ability to spontaneously form regular layers in solution and on solid supports (24). S-layers have been shown to have roles in the determination and maintenance of cell shape as virulence factors, as mediators of cell adhesion, and as regulators of immature dendritic and T cells. Moreover, they can also function as a protective coat, molecular sieve, murein hydrolase, and ion trap (4,8,13,17,19,25,29).S-layer proteins have several properties that make them an attractive target for the development of nanobiotechnological applications both in vivo and in vitro. In particular, a high number of protein subunits are displayed at the bacterial cell surface. Moreover, the prot...

Lactobacillus surface layer proteins: structure, function and applications

“…The analysis, based on measuring the surface accessibilities of the residues when the protein is in a monomeric or self-assembled form, grouped the residues according to their locations within the polymerized S-layer structure: to those located in the interior of the subunit, to those on the outer surface of the polymerized protein layer, to those on the inner surface of the layer and to those likely located in the subunit–subunit interface and pore or inner surface of the layer. The results confirmed the two-domain structure of SlpA and revealed several sites of high surface accessibility (Vilen et al 2009). …”

“…Similarly, surface display of GFP in the S-layer proteins on chicken Lactobacillus isolates has been achieved by utilizing the gene fragment encompassing the expression and secretion signals and the region encoding the cell wall binding domain of the S-layer protein of L. crispatus (Mota et al 2006). As a prerequisite for hybrid S-layer-based vaccine development, a systematic mapping of surface-accessible amino acids has been performed for the S-layer protein of L. brevis ATCC 8287 (Vilen et al 2009). Apart from hybrid S-layer proteins, non-adhesive antigen delivery vehicles like lactococci have been rendered adhesive by the surface display of adhesive S-layer proteins or S-layer-derived peptides such as those of L. crispatus JCM 5810 (Martinez et al 2000) or L. brevis ATCC 8287 (Åvall-Jääskeläinen et al 2003).…”

The Chloroplast Twin Arginine Transport (Tat) Component, Tha4, Undergoes Conformational Changes Leading to Tat Protein Transport

“…SA binding is easily scored by a large band shift of the target protein on SDS-PAGE. Br-PEG was used to label stroma-accessible sulfhydryl groups directly because it is unable to cross the membrane (24) and produces a detectible band shift of the target protein on SDS-PAGE.…”

The extracellular biology of the lactobacilli

“…In recent years, a number of S-layer proteins have been identified experimentally in L. acidophilus (especially the major S-layer protein SlpA), L. helveticus and L. brevis (Avall-Jääskeläinen & Palva, 2005;Hollmann et al, 2007;Goh et al, 2009;Vilen et al, 2009). The Pfam database contains different HMMs that correspond to S-layer protein domains responsible for noncovalent anchoring to the cell wall (SLAP or PF03217, SLH or PF00395, S_layer_C or PF05124 and S_layer_N or PF05123).…”