Structural and biochemical analyses of the Streptococcus pneumonia L,D-carboxypeptidase DacB.

The L,D-carboxypeptidase DacB plays a key role in the remodelling of Streptococcus pneumoniae peptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Šresolution crystal structure of DacB from S. pneumoniae TIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB from S. pneumoniae D39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, with Km and kcat values of 2.84 ± 0.37 mM and 91.49 ± 0.05 s(-1), respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

Structure of a novel O-linked N-acetyl-D-glucosamine (O-GlcNAc) transferase, GtfA, reveals insights into the glycosylation of pneumococcal serine-rich repeat adhesins.

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a ß-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides.

Structural insights into SraP-mediated Staphylococcus aureus adhesion to host cells.

Staphylococcus 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 platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å 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 ß-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP 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 host-pathogen interaction of S. aureus.

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC.

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/ß/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes themultiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr94 in OpuCC compared with Asp96 in OpuBC.

Crystal structure of the mucin-binding domain of Spr1345 from Streptococcus pneumoniae.

The surface protein Spr1345 from Streptococcus pneumoniae R6 is a 22-kDa mucin-binding protein (MucBP) involved in adherence and colonization of the human lung and respiratory tract. It is composed of a mucin-binding domain (MucBD) and a proline-rich domain (PRD) followed by an LPxTG motif, which is recognized and cleaved by sortase, resulting in a mature form of 171 residues (MF171) that is anchored to the cell wall. We found that the MucBD alone possesses comparable in vitro mucin-binding affinity to the mature form, and can be specifically enriched at the surface of human lung carcinoma A549 cells. Using single-wavelength anomalous dispersion (SAD) phasing method with the iodine signals, we solved the crystal structure of the MucBD at 2.0Å resolution, the first structure of MucBDs from pathogenic bacteria. The overall structure adopts an immunoglobulin-like fold with an elongated rod-like shape, composed of six anti-parallel ß-strands and a long loop. Structural comparison suggested that the conserved C-terminal moiety may participate in the recognition of mucins. These findings provided structural insights into host-pathogen interaction mediated by mucins, which might be useful for designing novel vaccines and antibiotic drugs against human diseases caused by pneumococci.