Structural basis for the peptidoglycan recognition by Helicobacter pylori Csd4

Helicobacter pylori infection causes a variety of gastrointestinal diseases including peptic ulcers and gastric cancer. The colonization of this bacterium in the gastric mucosa is required for the survival in the stomach. Its colonization of the gastric mucosa of human stomach depends on its motility, which is facilitated by the helical cell shape. In H. pylori, crosslinking relaxation or trimming of peptidoglycan muropeptide affects the helical shape. Among several cell shape-determining peptidoglycan hydrolases identified in H. pylori, Csd4 is a Zn2+-dependent D,L-carboxypeptidase that cleaves the bond between the γ-D-Glu and mDAP bond of the uncrosslinked tripeptide of peptidoglycan (L-Ala-γ-D-Glu-mDAP) to produce L-Ala-γ-D-Glu dipeptide and mDAP, promoting the helical cell shape. Inhibition of D,L-carboxypeptidase activity of Csd4 may represent a novel therapeutic approach. We report here the crystal structures of H. pylori Csd4 in three different states: the ligand-free form, the substrate-bound form, and the product-bound form. H. pylori Csd4 consists of three domains: an N-terminal D,L-carboxypeptidase domain, a novel β-barrel domain, and a C-terminal immunoglobulin-like domain. Our ligand-bound structures provide structural basis of peptidoglycan recognition by D,L-carboxypeptidase. H. pylori Csd4 recognizes primarily the terminal mDAP of the tripeptide substrate and undergoes a significant structural change upon binding either mDAP or mDAP-containing tripeptide.

Crystal structure of JHP933 from Helicobacter pylori J99

Helicobacter pylori infection is the main cause of chronic gastritis, gastric mucosal atrophy, peptic ulcer, and some forms of gastric cancer. There has been considerable interest in strain-specific genes found outside of the cag pathogenicity island, especially genes in the plasticity regions of H. pylori. In H. pylori strain J99, the plasticity region contains 48 genes ranging from jhp0914 to jhp0961. Because little is known about many of these genes in the plasticity region, further studies are necessary to elucidate their roles in H. pylori-associated pathogenesis. The JHP933 protein, encoded by the jhp0933 gene in the plasticity region of H. pylori J99, is one of the prevalently expressed proteins in some gastritis and peptic ulcer patients. However, its structure and function remain unknown. Here, we have determined the crystal structure of JHP933, revealing the first two-domain architecture of DUF1814 family. The N-terminal domain has the nucleotidyltransferase fold and the C-terminal domain is a helix bundle. Structural similarity of JHP933 to known nucleotidyltransferases is very remote, suggesting that it may function as a novel nucleotidyltransferase. It is expected that this study will facilitate functional characterization of JHP933 to obtain an insight into its role in pathogenesis by the H. pylori plasticity region.

Crystal structure of PhoU from Pseudomonas aeruginosa

The PhoU protein in bacteria plays a role in maintaining phosphate homeostasis by regulating the Pho regulon. Recent studies showed that PhoU is essential for normal growth and is also involved in persister formation. PhoU is a potential target for overcoming drug tolerance of pathogenic bacteria. However, the exact mechanism of PhoU functions is still unknown. Here we have determined the crystal structure of PhoU from Pseudomonas aeruginosa at 2.28 Å resolution by Se SAD method. P. aeruginosa PhoU exists as a dimer in the crystals. A monomer of P. aeruginosa PhoU consists of six alpha-helices, which form two similar helical bundles. P. aeruginosa PhoU shares four conserved sequence motifs. Interestingly, P. aeruginosa PhoU has distinct features in some loops and the surface charge distribution. Two monomers of P. aeruginosa PhoU dimerize in a slightly different manner to those of other PhoU proteins. The present structure of PhoU from a bacterial pathogen may be useful for the antibacterial drug discovery.

Crystal structure of the Csd3 protein from Helicobacter pylori

The helical cell shape of Helicobacter pylori facilitates the penetration of thick gastric mucus and promotes virulence. The peptidoglycan plays a structural role in the bacterial cell wall and its controlled modification is essential for determining the helical shape. Several H. pylori genes were identified to contribute to its helical cell shape through alterations in peptidoglycan crosslinking and trimming of the peptide (Sycuro et al., 2010; Sycuro et al., 2012). One of them is the hp0506 gene that encodes a putative periplasmic peptidase belonging to the M23-family of zinc-metallopeptidase (Sycuro et al., 2010). The HP0506 protein carries out not only a D,D-endopeptidase activity but also a D,D-carboxypeptidase activity. Hence, it has been named Helicobacter D,D-peptidase A (HdpA) and cell shape determinant 3 (Csd3). Csd3 is the first enzyme belonging to the M23-peptidase family that can perform the D,D-carboxypeptidation to regulate the cell shape (Mathilde et al., 2010). To gain structural and functional insights at the molecular level, we have determined the crystal structure of Csd3 at 2.1 Å resolution by using the Pt SAD data. H. pylori Csd3 consists of three domains including a LytM domain, which contains the highly conserved active site motif among the M23 metallopeptidase family. An anomalous scattering experiment with Zn2+ confirmed the metal-binding site in the active site. The Zn2+ ion is tetrahedrally coordinated and a catalytic water for peptide hydrolysis is absent in the active site of Csd3. Furthermore, domain 1 blocks the active site, thus prohibiting the substrate peptide binding. Our mass analysis shows that the full-length Csd3 is inactive as the D,D-carboxypeptidase. These results suggest that proteolytic processing may be necessary for the activation of Csd3.

Crystal structure of Rv2258c, a putative SAM-dependent methyltransferase

The Mycobacterium tuberculosis protein Rv2258c belongs to a large family of putative S-adenosylmethionine (SAM)-dependent methyltransferases in mycobacteria. As part of a laboratory-scale structural genomics project on conserved hypothetical proteins in pathogenic bacteria, we have determined the crystal structure of Rv2258c from M. tuberculosis H37Rv at 1.8 Å resolution. The crystals of apo Rv2258c belong to space group C2, with unit cell parameters a = 109.2 Å, b = 140.6 Å, c = 97.2 Å, and β = 98.5⁰. Assuming that three monomers are present in the asymmetric unit, the Matthews parameter and the solvent fraction are 3.32 Å3 Da-1 and 63.0%, respectively. The crystal structure of Rv2258c has been solved by a combination of the molecular replacement and single wavelength anomalous diffraction technique, using platinum atoms as anomalous scattering centers and the coordinates of the RebM from Lechevalieria aerocolonigenes (PDB entry 3BUS) as the search model. In addition, two binary complex structures, Rv2258c-SAM and Rv2258c-S-adenosylhomocysteine (SAH), have been determined by molecular replacement using the model of apo Rv2258c.

Structure of STM3169, a tripartite ATP-independent periplasmic transporter

Substrate-binding proteins (SBPs) form a group of proteins that are commonly related to membrane protein complexes for transport or cell signal transduction. SBPs are comprised of prokaryotic ATP-binding cassette (ABC)-transporters, prokaryotic tripartite ATP-independent periplasmic (TRAP)-transporters, prokaryotic two-component regulatory systems, eukaryotic guanylatecyclase-atrial natriuretic peptide receptors, G-protein coupled receptors (GPCRs) and ligand-gated ion channels (Berntsson et al., 2010).The TRAP transporters are less known as compared with ABC transporters but are ubiquitous in prokaryotes. The TRAP transporters are important elements of solute uptake systems in prokaryotes. These transporters contain two membrane protein components, predicted to have four and twelve transmembrane helices, respectively. In the TRAP transporters of DctP-type, substrate recognition is mediated through a well-conserved and specific arginine/carboxylate interaction in the SBP (Mulligan et al., 2011). Here we have determined the crystal structure of the TRAP transporter from Salmonella entericaserovarTyphimurium. Unexpectedly, this structure shows that various ligands can bind to the TRAP transporters. It provides insights into substrate binding mechanism in the TRAP transporter system.

Crystal structure of Msm Eis bound with paromomycin

The Rv2416c gene of Mycobacterium tuberculosis (Mtb) encodes the enhanced intracellular survival (Eis) protein that enhances intracellular survival of the pathogen in host macrophages during infection. The Eis protein is secreted by Mtb into the cytoplasm of the phagocyte during intracellular infection and modulates the host immune response. It was shown to confer resistance to aminoglycosides by acetylation. Interestingly, nonpathogenic Mycobacterium smegmatis (Msm) contains a homologous eis gene (MSMEG_3513) that encodes a homolog of Mtb Eis. We discovered that Mtb Eis is an Nε-acetyltransferase, acetylating Lys55 of DUSP16/MKP-7, a JNK-specific phosphatase, whereas Msm Eis is an Nα-acetyltransferase (Kim et al., 2012). We also reported that Msm Eis acetylated the aminoglycosides as quickly as, or more rapidly than, Mtb Eis (Kim et al., 2012). Here we present the crystal structure of Msm Eis in the paromomycin-bound form. This structure provides a detailed structural view of the interactions between paromomycin and Msm Eis. Glu137 and Gly401 of Msm Eis are key residues that interact with paromomycin. Our work may facilitate a structure-guided discovery of Mtb Eis inhibitors as a novel anti-TB drug.