Structural And Functional Insights Into Staphylococcus Aureus Ligand Binding Region Of SraP And Streptococcus Pneumoniae L,D-Carboxypeptidase DacB

(Ⅰ) Structure and function of ligand binding region of SraPStaphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelet. It remains unclear how SraP interacts with human host. Here we report the2.05A crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differing in calcium coordination pattern. Small-angle X-ray scattering and molecular dynamic simulation indicated the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outward. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP specifically binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated staphylococcal infection. (Ⅱ) Structure and function of L,D-carboxypeptidase DacBStreptococcus pneumoniae DacB is an L,D-carboxypeptidase hydrolyzing the peptide bond between L-Lys and D-Ala in pneumococcal peptidoglycan (PG). Deletion of the dacB gene has been shown to result in defects in cell shape and septation implying its important role in peptidoglycan remodeling. Therefore, DacB represents a promising target for the development of new types of antibiotics. However, the structure fold and catalytic mechanism of this enzyme remians unknown. Here, we determined the high-resolution structure of DacB in complex with acetate, representing the first Gram-positive bacterial L,D-carboxypeptidase structure. DacB adopts a globular fold, with several a-helices surrounding a central β-sheet. Structure analysis of DacB reveals a new member of the M15B peptidase family. The active site contains an unexpected six-coordinate zinc ion with a His-Asp-His-Glu tetrad and two metal-bound water molecules. We also identified that the tetrapeptide (Ala-iGln-L-Lys-D-Ala) can be used as a favorite substrate by the enzyme. Molecular docking and digestion assays with the tetrapeptide allowed us to identify key residues in ligand binding and catalysis. Based on these findings,we could assign DacB and other L,D-carboxypeptidases in Gram-positive bacteria as metalloenzymes and proposed the identification of specific inhibitors of this important class of enzymes according to a putative catalytic mechanism.