Hai Pang

The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiological agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, we report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to our knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.

Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

Background It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined. Methodology/Principal Findings We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C2–C8), and its suitable substrate was p-nitrophenyl caproate (C6) with optimal catalytic conditions of 39°C and pH 8.0. Conclusions/Significance Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.

Crystal structure of the pyridoxal-5′-phosphate-dependent serine dehydratase from human liver

L‐serine dehydratase (SDH), a member of the β‐family of pyridoxal phosphate‐dependent (PLP) enzymes, catalyzes the deamination of L‐serine and L‐threonine to yield pyruvate or 2‐oxobutyrate. The crystal structure of L‐serine dehydratase from human liver (hSDH) has been solved at 2.5 Å‐resolution by molecular replacement. The structure is a homodimer and reveals a fold typical for β‐family PLP‐dependent enzymes. Each monomer serves as an active unit and is subdivided into two distinct domains: a small domain and a PLP‐binding domain that covalently anchors the cofactor. Both domains show the typical open α/β architecture of PLP enzymes. Comparison with the rSDH‐(PLP‐OMS) holo‐enzyme reveals a large structural difference in active sites caused by the artifical O‐methylserine. Furthermore, the activity of hSDH‐PLP was assayed and it proved to show catalytic activity. That suggests that the structure of hSDH‐PLP is the first structure of the active natural holo‐SDH.

Crystal Structure of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of α/β hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the α/β fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis.

Expression, Purification, Crystallization and Preliminary X-Ray Analysis of Human Spindlin1, an Ovarian Cancer-Related Protein

Human spindlin1 is a newly screened and identified gene product related to ovarian carcinomas and is highly homologous to mouse spindlin. It is an abundant maternal transcript expressed in the mouse during the transition from oocyte to embryo. Here, the recombinant human spindlin1 has been overexpressed in Escherichia coli BL21, purified and crystallized using the hanging-drop vapour-diffusion method. Crystals diffracting to 2.25 A resolution were obtained using ammonium sulfate as precipitant. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a =40.7 A, b =84.4 A, c =136.4 A, alpha=beta=gamma=90 degrees . Assuming two molecules per asymmetric unit, the solvent content is calculated to be 42.4%.

Crystallization and preliminary crystallographic analysis of human serine dehydratase

L-Serine dehydratase (SDH) catalyzes the pyridoxal phosphate (PLP) dependent deamination of L-serine to yield pyruvate. Recombinant human serine dehydratase was crystallized by the hanging-drop vapour-diffusion method. Crystals were grown at 291 K using (NH4)(2)SO4 as precipitant. Diffraction data were obtained to a resolution of 2.5 A from a single frozen crystal using Cu Kalpha radiation. The crystal belongs to space group I422, with unit-cell parameters a = 157.4, b = 157.4, c = 59.2 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains one molecule and has a solvent content of about 46%.

Crystallization and preliminary crystallographic analysis of RSB-66, a novel round spermatid-specific protein

Crystals of the RSB-66 protein have been grown at 291 K using NaCl as precipitant. In the refinement of the crystallization this protein, the crystallographic PCR method was used and was found to help in obtaining the best crystals more quickly and easily. The diffraction pattern of the crystal extends to 2.7 A resolution in-house. A full set of X-ray diffraction data were collected to 2.7 A from a single crystal. The crystals belong to space group P4212, with unit-cell parameters a = 90.4, b = 90.4, c = 122.2 A, alpha = beta = gamma = 90 degrees. The presence of two or three molecules per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.22 or 2.14 A(3) Da(-1), respectively.

Myricetin ameliorates the symptoms of collagen-induced arthritis in mice by inhibiting cathepsin K activity

Abstract Myricetin, a common dietary flavonoid, is widely distributed in fruits and vegetables. It is known to be a food supplement contributing to human health because of its immune modulatory function, and its antioxidation, antitumor, and anti-inflammatory properties. In the present study, myricetin was shown to directly inhibit cathepsin K activity, a highly potent collagenase, which is the predominant papain-like cysteine protease expressed in osteoclasts and synovial fibroblasts. It was shown that the IC50 of myricetin for the recombinant human cathepsin was 585.3 µmol/L. Also, myricetin proved to have positive effects in murine collagen-induced arthritis (CIA). Mice suffering from CIA received a daily dose of myricetin (25 mg/kg, per os). During the study, the clinical severity of the CIA and the histopathology were evaluated. Biomarkers related to the histological evaluation of cartilage degradation, namely deoxypyridinoline, cartilage oligomeric matrix protein and C-terminal telopeptide degradation product of type I collagen (CTX-I), were analyzed. Myricetin treatment reduced the levels of biomarkers indicative of cartilage degradation (p < 0.05) and ameliorated the symptoms of CIA in mice at the clinical level (p < 0.01). As the inhibitory effect of myricetin on cathepsin K activity induced beneficial effects on CIA in mice, further investigation of therapeutic interventions with myricetin in other mammals or in human rheumatoid arthritis is recommended.

Purification, crystallization and preliminary X-ray diffraction analysis of human enolase-phosphatase E1.

Enolase-phosphatase E1 (MASA) is a bifunctional enzyme in the ubiquitous methionine-salvage pathway and catalyzes the continuous reaction of 2,3-diketo-5-methylthio-1-phosphopentane to yield the acireductone metabolite. Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Diffraction data were collected to 1.7 A resolution from SeMet-derivative crystals at 100 K using synchrotron radiation. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 54.02, b = 57.55, c = 87.32 A. The structure was subsequently solved by the multi-wavelength anomalous diffraction (MAD) phasing method.

Purification, crystallization and preliminary X-ray analysis of human pirin

Pirin is a novel highly conserved nuclear protein, but very little is known about its cellular function. Human pirin has been cloned, expressed, purified and crystallized using PEG as precipitant. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 42.3, b = 67.0, c = 107.3 A, alpha = beta = gamma = 90 A. It contains one molecule per asymmetric unit and diffracts to 2.0 A under cryoconditions (100 K) using an in-house Cu rotating-anode X-ray generator.