| 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 protein subunits are able to spontaneously self-assemble into a regularly arranged lattice structure both in solution and on solid supports (1, 27, 30, 31). However, despite the high prevalence of S-layers in nature, their molecular structure remains poorly elucidated. In particular, knowledge about the spatial organization of amino acid residues in S-layer proteins or the interactions between these residues and other subunits is limited. The poor solubility of protein assemblies and the absence of stoichiometrically defined oligomers have hindered attempts to apply nuclear magnetic resonance or hydrogen/deuterium exchange mass spectroscopy. In addition, the intrinsic property of S-layer proteins to form two-dimensional lattices has hampered efforts to obtain three-dimensional crystals required for X-ray crystallography (12, 31). To our knowledge, only part of the structure of one S-layer protein, SbsC of Geobacillus stearothermophilus, has been determined by X-ray crystallography (18). Since high-resolution, three-dimensional structural data are mostly lacking, traditional mutation-based techniques are presently the methods of choice. In cysteine-scanning mutagenesis (CSM), a series of mutant proteins is generated by replacing single residues with cysteine, which contains a sulfhydryl group amenable to further chemical modification. The spatial locations of amino acid residues within the S-layer protein SbsB of gram-positive thermophile G. stearothermophilus PV72/p2 have been analyzed by CSM. A total of 75 residues out of 920 were studied, identifying 23 residues located at the surface of protein monomers, five of those located on the outer surface of the protein lattice (10). These mutant proteins were subsequently analyzed by a cross-linking screen to assess residues accessible in monomeric form to the protein/protein interface and the inner surface of the lattice (12).In the genus Lactobacillus, S-layers have been found in several species. S-layer protein genes have been sequenced from L. brevis, L. helveticus, and L. acidophilus group organisms. Sequence similarity between Lactobacillus S-layer protein genes can be found only between closely related Lactobacillus species. Therefore, the primary sequences of Lactobacillus S-layer proteins show extensive variability, with the number of identical amino acids varying from 7 to 100% between different proteins. As a group, Lactobacillus S-layer proteins differ from those of most other bacteria in their smaller sizes (25 to 71 kDa) and higher calculated isoelectric point (pI) values (9.4 to 10.4) (1). The presence of two or more S-layer protein genes in the same strain is common in lactobacilli (5, 6, 11, 28, 35); however, only one S-layer protein gene, slpA, has so far been described to be present in the genome of L. brevis ATCC 8287. SlpA is a 435-amino-acid, 46-kDa S-layer protein that assembles into a lattice of oblique symmetry on the bacterial surface (2, 36). L. brevis ATCC 8287 has GRAS (generally recognized as safe) status and has been shown to possess probiotic properties (21), which make SlpA a very attractive subject, e.g., in the development of live oral vaccines. Moreover, a recent report using differential scanning calorimetry suggests that in comparison with other S-layer proteins, SlpA is resistant to high temperatures (21). This thermal stability could prove potentially useful in a variety of in vitro S-layer applications currently being planned or under development (27, 30, 31). Recently, SlpA was characterized to consist of an N-terminal cell wall binding domain (residues 1 to 178) and a C-terminal self-assembly domain (179 to 435) (3). For the development of applications that take advantage of these characteristics, further investigation of SlpA at the molecular level is essential.Herein, we use CSM and targeted chemical modification to assign 46 amino acid residues of SlpA to spatial locations in the protein monomer and in the lattice according to their surface accessibility. We focused mainly on the self-assembly domain, the region facing the outer surface of the protein lattice and thus most amenable to insertions and chemical modification. Two different marker molecules were used to modify cysteine-containing mutant proteins that were either in solution or attached to the cell wall. The results were subsequently evaluated taking advantage of the recent new information on SlpA domain boundaries (3). We were able to distinguish residues located in the outer and inner surfaces of the lattice, protein interior, and interface/pore regions. The information gathered here can be used in the development of further biotechnological and nanobiological applications, both in vitro and in vivo, that benefit from a thermostable S-layer protein from a GRAS bacterium with health-beneficial properties. |
