SAP_SLH

The Surface Layer Homology (SLH) Domain of Sap from Bacillus Anthracis (PDB ID: 6BT4) is responsible for the incorporation of chimeric proteins into and the formation of the Surface Layer (S-layer) of the bacteria (1). The SLH domain of SAP is able to do this by binding to secondary cell wall polysaccharides (SCWP) that extend from the from the cell wall (1). It is these S-layer proteins that are the key virulence for this class of proteins and diseases (2). Virulence factors are proteins and other molecules produced and secreted by pathogens (bacteria, protozoa, viruses, and, fungi) that add to the organism?s effectiveness. The SLH domain of Sap is composed of a single subunit and three subdomains (3).

Crystals of the SLH Domain of Sap were obtained by the following process. B. Anthracis was grown in culture overnight. Next, the resulting culture was centrifuged at 10,000 x g, and following sediment was suspended in 3 M urea and then heated in a boiling water bath for thirty minutes (1). The solution was washed in water followed by a rinsing in a phosphate buffered saline (PBS) solution (1). The SLH domain of Sap crystal structure is missing seven residues (57-63).

The molecular weight of the SLH domain of Sap is 22868.2 Da and the isoelectric point is 6.5 using (3, 4). The primary structure of the protein is composed of roughly one third ionic residues, one fourth polar uncharged residues and two fifths unipolar residues (1). In bacterial S-layer SLH domains there is a partially conserved Ile-Thr-Arg-Ala-Glu motif. In B Anthracis that motif has the sequences Leu-Thr-Arg-Ala-Glu, Ile-Asp-Arg-Val-Ser, and Val-Thr-Lys-Ala-Glu for SLH1, SLH2 and SLH¬3 respectively. This motif appears in the last four residues of the B loop and the first residue of their respective central alpha helix. The secondary structure of the SLH domain of Sap is composed of 37% alpha helices (9 helices; 75 residues) and 2% beta sheets (6 strands; 6 residues) (3). Three of the alpha helices (referred to as helices a, b, and c) are centrally located, parallel to each other, and point out of the place of the macromolecule. Each of these three alpha helices have hydrophobic facing the center and uncharged polar residues facing away. Surrounding these three parallel alpha helices are three more alpha helices roughly perpendicular to the first three (referred to as helices x, y, and z) (1). The SLH domain of Sap has three sub domains including: SLH1 (helices c and x, residues 32-90), SLH2 (helices a and z, residues 91-151) and SLH3 (helices b and y, residues 152-209) (1). Together these three subdomains form a trefoil shape, with each of the subdomains donating an alpha helix to the center three helix bundle (1). The clefts that form between the outer prongs of the protein are known as the interprong grooves (IPGs) (5). There are very little beta pleated sheets present in the protein, but there are significant sections of random coils, which contribute to the binding of the SCWP units in the IPGs.

There are two known ligands for the SLH Domain of Sap from B. Anthracis: Sulfate ions (SO4-2) and 2-(acetylamino)-4-O-{2-(acetylamino)-4,6-O-[(1S)-1-carboxyethylidene]-2-deoxy-beta-D-mannopyranosyl}-2-deoxy-beta-D-glucopyranose (a pyruvylated SCWP known as KMP). The protein holds the sulfate ion by utilizing non-covalent interactions, namely hydrogen bonds between the ion and several charged amino acids on the nearby alpha helix, including: Arg-131, Ser-178, Val-179, Gly-180, Thr-181, Trp-185 and Phe-1997 (3). The function of this sulfate ion is to allow the IPG of the protein to fit more snuggly around the SCWP, making the interaction between the two stronger, meaning more stable and more favorable interaction. The second ligand is the SCWP known as KMP. KMP is part of a repeating polysaccharide structure that extends out from the cell and the binding of SAP-SLH to this ligand is what attaches it to the cell and begins the formation of the S-layer. KMP is also non-covalently bonded to the protein via hydrogen bonds formed by polar amino acids, and also through non-polar Van Der Waals interactions (3). The amino acids that form these hydrogen bonds and Van Der Waals forces are the following: Arg-72, Phe-123, Ser-136, Trp-162, Lys-168 (3).

Proteins with similar structures to the SLH Domains of Sap from B. Anthracis were identified and compared with the SLH domain of Sap by primarily two sources: The Position Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST), and The Dali Server. PSI-Blast compares two proteins based on their primary structure or sequence, and from this comparison, assigns an E value for the two proteins. An E value of 0.5 or below indicates a high level of sequential similarity between the two primary sequences. The Dali Server makes comparisons based on a proteins tertiary structure, and based on the similarities between the two structures assigns a Z-score. A Z-score of 2.0 or above indicates a protein with high similarities in their tertiary structure.

SpaA-SLH Domains of Paenibacillus Alvei (PDB ID: 6CWC) was selected for comparison with the SLH Domain of Sap from B. Anthracis for several reasons. First, it has very similar primary sequence and tertiary structures. SpaA-SLH, in comparison with Sap-SLH has an E score of 3.0 x 10-8 and a Z score of 19.1 (6, 7). The tertiary structure of the two proteins is very similar as the function of SpaA-SLH is, just like SAP-SLH, is form the S-layer of Gram-positive bacteria by attaching to SCWP (8). SpaA-SLH is a single monomer composed of three subdomains formed into a trefoil shape. While a similar tertiary structure is to be expected for proteins performing the same function, one would expect to see more differences in primary structure given the two proteins from different organisms in different genera. The low E-scores shows that the protein that performs this function has been conserved throughout the divergence of these two species. The only major difference is the method by which the protein binds to the SCWP.

The S-Layer of bacteria is found in almost all archaea and encloses the entirety of the cell. The S-layer is the part of the bacteria that interacts with the environment and as such must provide a wide variety of functions. Such functions include, but are not limited to: protection against bacteriophages, immunoevasion and immunosuppression of the host's immune response and obtaining nutrients. These factors are specifically known as virulence factors which are molecules that allow bacteria to grow, reproduce and evade the body's natural response to foreign organisms. Bacterial infections cause twenty-three thousand deaths per year in hospitals and it is the bacteria's S-Layer that allow them to evade the immune system and antibiotics. Because S-Layer proteins and the associated virulence factors are present in almost all eubacteria and archaebacteria, research into their identification and assembly could lead to decreased immunoevasion by the development of drugs that allow the body's immune system to identify and destroy bacteria.