Deqiang Wang

Structural Basis of the Novel S. pneumoniae Virulence Factor, GHIP, a Glycosyl Hydrolase 25 Participating in Host-Cell Invasion

Pathogenic bacteria produce a wide variety of virulence factors that are considered to be potential antibiotic targets. In this study, we report the crystal structure of a novel S. pneumoniae virulence factor, GHIP, which is a streptococcus-specific glycosyl hydrolase. This novel structure exhibits an α/β-barrel fold that slightly differs from other characterized hydrolases. The GHIP active site, located at the negatively charged groove in the barrel, is very similar to the active site in known peptidoglycan hydrolases. Functionally, GHIP exhibited weak enzymatic activity to hydrolyze the PNP-(GlcNAc)5 peptidoglycan by the general acid/base catalytic mechanism. Animal experiments demonstrated a marked attenuation of S. pneumoniae-mediated virulence in mice infected by ΔGHIP-deficient strains, suggesting that GHIP functions as a novel S. pneumoniae virulence factor. Furthermore, GHIP participates in allowing S. pneumoniae to colonize the nasopharynx and invade host epithelial cells. Taken together, these findings suggest that GHIP can potentially serve as an antibiotic target to effectively treat streptococcus-mediated infection.

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein-protein interfaces.

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow-brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein-protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.

Purification, crystallization and preliminary X-ray studies of the putative lysozyme SP0987 from Streptococcus pneumoniae.

Streptococcus pneumoniae SP0987, which was identified as a hypothetical protein, has a very low sequence identity to other well characterized lysozyme structures. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography has been used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of SP0987 from Streptococcus pneumoniae TIGR4 are reported. The crystal belonged to space group P2(1)2(1)2(1) (with unit-cell parameters a = 36.46, b = 40.89, c = 147.44 A) and diffracted to a resolution of 1.85 A. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 2.02 A(3) Da(-1).

Cloning, purification, crystallization and preliminary X-ray studies of flagellar hook scaffolding protein FlgD from Pseudomonas aeruginosa PAO1.

FlgD regulates the assembly of the hook cap structure to prevent leakage of hook monomers into the medium and hook monomer polymerization and also plays a role in determination of the correct hook length, with the help of the FliK protein. In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. The crystal belonged to space group I222 and diffracted to a resolution of 2.5 A, with unit-cell parameters a = 116.47, b = 118.71, c = 118.85 A. The crystals are most likely to contain three molecules in the asymmetric unit, with a V(M) value of 2.73 A(3) Da(-1).

Mechanism of the allosteric regulation of Streptococcus mutans 2'-deoxycytidylate deaminase

In cells, dUMP is the intermediate precursor of dTTP in its synthesis during deoxynucleotide metabolism. In Gram-positive bacteria and eukaryotes, zinc-dependent deoxycytidylate deaminases (dCDs) catalyze the conversion of dCMP to dUMP. The activity of dCD is allosterically activated by dCTP and inhibited by dTTP. Here, the crystal structure of Streptococcus mutans dCD (SmdCD) complexed with dTTP is presented at 2.35 Å resolution, thereby solving the first pair of activator-bound and inhibitor-bound structures from the same species to provide a more definitive description of the allosteric mechanism. In contrast to the dTTP-bound dCD from the bacteriophage S-TIM5 (S-TIM5-dCD), dTTP-bound SmdCD adopts an inactive conformation similar to the apo form. A structural comparison suggests that the distinct orientations of the triphosphate group in S-TIM5-dCD and SmdCD are a result of the varying protein binding environment. In addition, calorimetric data establish that the modulators bound to dCD can be mutually competitively replaced. The results reveal the mechanism underlying its regulator-specific activity and might greatly enhance the understanding of the allosteric regulation of other dCDs.

Crystallization and preliminary X-ray crystallographic studies of DnaJ from Streptococcus pneumoniae

DnaJ, cooperating with DnaK and GrpE, promotes the folding of unfolded hydrophobic polypeptides, dissociates protein complexes and translocates protein across membranes. Additionally, DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. The crystals belong to space groups I222 or I2₁2₁2₁ and the diffraction resolution is 3.0 Å with unit-cell parameters a=47.68, b=104.45, c=234.57 Å. The crystal most likely contains one molecule in the asymmetric unit, with a VM value of 3.24 Å3 Da(-1) and a solvent content of 62.1%.

Cloning, purification, crystallization and preliminary X-ray studies of human α1-microglobulin.

α(1)-Microglobulin (α(1)m) is one of the phylogenetically most widespread lipocalins and is distributed in various organs and tissues, including liver, heart, eye, kidney, brain, lung, pancreas and skeletal muscle. α(1)m has been found to exert multifarious functions, including interacting with IgA, albumin and prothrombin, binding strongly to haem and exhibiting reductase activity. Nevertheless, little structural information is available regarding these functions of α(1)m. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography was used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of human α(1)m are reported. The crystal belonged to space group P4(3), with unit-cell parameters a = b = 36.45, c = 112.68 Å, and diffracted to a resolution of 2.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 1.63 Å(3) Da(-1).

Crystal Structure of an Invasivity-Associated Domain of SdrE in S. aureus

The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with β2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes.

Cloning, purification, crystallization and preliminary X-ray studies of HMO2 from Saccharomyces cerevisiae

The high-mobility group protein (HMO2) of Saccharomyces cerevisiae is a component of the chromatin-remodelling complex INO80, which is involved in double-strand break (DSB) repair. HMO2 can also bind DNA to protect it from exonucleolytic cleavage. Nevertheless, little structural information is available regarding these functions of HMO2. Since determination of three-dimensional structure is a powerful means to facilitate functional characterization, X-ray crystallography has been used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of HMO2 from S. cerevisiae are reported. The crystal belonged to space group P222, with unit-cell parameters a = 39.35, b = 75.69, c = 108.03 Å, and diffracted to a resolution of 3.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a VM value of 3.19 Å(3) Da(-1).

Purification, crystallization and preliminary X-ray crystallographic study of the tRNA pseudouridine synthase TruB from Streptococcus pneumoniae.

The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine (Ψ) at residue 55 of elongator tRNAs. In order to better elucidate the functions of TruB in the formation of pseudouridine, the three-dimensional structure of full-length TruB was determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported. The crystal belonged to space group P2, with unit-cell parameters a = 37.65, b = 78.09, c = 56.33 Å, β = 102.05°, and diffracted to a resolution of 1.7 Å. The crystal is most likely to contain one molecule in the asymmetric unit, with a VM value of 2.40 Å(3) Da(-1).