Biomedical subjects

Quan Hao

Publications and source records attributed to Quan Hao.

29 records · Page 2Linked to original sources

PubMed2004

Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitor-like fold.

Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-angstrom resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N-terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family.

Amino Acid Sequence↗

PubMed2004

Purification, characterization, crystallization and preliminary X-ray crystallographic analysis of two novel C-type lectin-like proteins: Aall-A and Aall-B from Deinagkistrodon acutus venom.

Aall-A and Aall-B, two novel heterodimeric snake-venom C-type lectin-like proteins (sv-CLPs), were purified from the venom of Deinagkistrodon acutus from Anhui, China. Strikingly, both these proteins can localize on and congregate human erythrocytes, instead of aiming at the common targets of sv-CLPs such as platelet glycoproteins, von Willebrand factors, coagulant factors etc. The crystals of Aall-A belong to space group P2, with unit-cell parameters a = 105.2, b = 56.2, c = 108.7 A, beta = 100.5 degrees , and diffract to 2.0 A resolution, while the crystals of Aall-B belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 36.8, b = 56.5, c = 149.2 A, and diffract to 2.2 A resolution. To our knowledge, this is the first report of sv-CLPs with this unique function and of their preliminary crystallographic analysis.

Amino Acid Sequence↗

PubMed2004

The atomic resolution crystal structure of atratoxin determined by single wavelength anomalous diffraction phasing.

By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.

Amino Acid Motifs↗

PubMed2004

Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine.

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.

6-Phytase↗

PubMed2004

Purification, partial characterization, crystallization and preliminary X-ray diffraction of two cysteine-rich secretory proteins from Naja atra and Trimeresurus stejnegeri venoms.

Cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals and snake venoms. They possess apparent homology but varying functions. The structure of CRISPs has remained elusive. Two novel members of the family, natrin and stecrisp, have been purified from Naja atra and Trimeresurus stejnegeri venoms, respectively. Their crystals diffract X-rays to resolution limits of 2.1 and 1.6 angstroms, respectively, and belong to the orthorhombic system with different space groups, unit-cell parameters and numbers of molecules per asymmetric unit. Their structures will contribute a structural basis for further functional studies of this family.

Amino Acid Sequence↗

PubMed2004

Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom.

A snake-venom protein named AHP-LAAO has been purified from Agkistrodon halys pallas venom using four-stage chromatography. AHP-LAAO is a novel member of the snake-venom L-amino-acid oxidase family. Its amino-acid sequence shows high homology to other members of this family. For L-leucine, the values of k(cat) and K(M) are 31.1 s(-1) and 0.25 mM, respectively. The molecular weight of AHP-LAAO is about 60.7 kDa as determined by MALDI-TOF mass spectrometry. AHP-LAAO can also induce apoptosis of cultured Hela cells. Two sets of diffraction data with similar resolution limits (about 2.5 A) were collected independently at MacCHESS (Cornell High Energy Synchrotron Source, USA) and IHEP (Institute of High Energy Physics, Beijing, China). The crystals belong to space group I2(1)3, with unit-cell parameter a = 169.31 A, corresponding to one molecule in the asymmetric unit and a volume-to-weight ratio of 3.33 A(3) Da(-1). The final structural model is similar to that of L-amino-acid oxidase from Calloselasma rhodostoma venom.

Amino Acid Oxidoreductases↗

PubMed2004

ADP-ribosyl cyclase; crystal structures reveal a covalent intermediate.

ADP-ribosyl cyclase catalyzes the elimination of nicotinamide from NAD and cyclization to cADPR, a known second messenger in cellular calcium signaling pathways. We have determined to 2.0 A resolution the structure of Aplysia cyclase with ribose-5-phosphate bound covalently at C3' and with the base exchange substrate (BES), pyridylcarbinol, bound to the active site. In addition, further refinement at 2.4 A resolution of the structure of nicotinamide-bound cyclase, which was previously reported, reveals that ribose-5-phosphate is also covalently bound in this structure, and a second nicotinamide site was identified. The structures of native and mutant Glu179Ala cyclase were also solved to 1.7 and 2.0 A respectively. It is proposed that the second nicotinamide site serves to promote cyclization by clearing the active site of the nicotinamide byproduct. Moreover, a ribosylation mechanism can be proposed in which the cyclization reaction proceeds through a covalently bound intermediate.

ADP-ribosyl Cyclase↗

PubMed2004

Crystallographic snapshots of Aspergillus fumigatus phytase, revealing its enzymatic dynamics.

Understanding of the atomic movements involved in an enzymatic reaction needs structural information on the active and inactive native enzyme molecules and on the enzyme-substrate, enzyme-intermediate, and enzyme-product(s) complexes. By using the X-ray crystallographic method, four crystal structures of Aspergillus fumigatus phytase were obtained at resolution higher than 1.7 A. The pH-dependent catalytic activity of A. fumigatus phytase was linked to three water molecules that may prevent the substrate from binding and thus block nucleophilic attack of the catalytic imidazole nitrogen. Comparison of various structures also identified the water molecule that attacks the phosphamide bond during the hydrolysis process, and established the hydrolysis pathway of the intermediate. Additionally, two reaction product phosphates were observed at the active site, suggesting a possible product release pathway after hydrolysis of the intermediate. These results can help explain the catalytic mechanism throughout the whole acid phosphatase family, as all key residues are conserved.

6-Phytase↗

PubMed2003

Purification, partial characterization and crystallization of acucetin, a protein containing both disintegrin-like and cysteine-rich domains released by auto-proteolysis of a P-III-type metalloproteinase AaH-IV from Agkistrodon acutus venom.

AaH-IV, a P-III-type metalloproteinase found in Agkistrodon acutus venom, readily cleaves itself to release a stable protein named acucetin at 310 K under neutral and weakly alkaline conditions. A partial amino-acid residue sequence of acucetin indicates that the protein has a high homology to snake-venom proteins containing both disintegrin-like and cysteine-rich domains. Acucetin has been crystallized in space group R32, with hexagonal unit-cell parameters a = b = 155.98, c = 76.07 A. The V(M) value of about 2.97 A(3) Da(-1) suggests the presence of only one molecule in the asymmetric unit.

Agkistrodon↗

PubMed2003

S-SWAT (softer single-wavelength anomalous technique): potential in high-throughput protein crystallography.

The drive for yet higher output of protein crystal structures sustains an interest in streamlining data-collection protocols. Highly organized automated robotics attend to one aspect. The data-collection elapsed time also depends on the number of wavelengths used, the data redundancy and the desired diffraction resolution limit. Synchrotron-radiation beamlines offer a centralized data-collection approach with considerable flexibility of choice of wavelength(s) with high beam intensity and fine collimation. The arrival of automated IPs (imaging plates) and CCDs (charge-coupled devices) with sample freezing has widened the scope for ease of data collection and achieving a high redundancy, the latter at the cost of elapsed time per sample. This paper reports a single-wavelength approach where a large optimized f" signal involving the Xe L1-absorption edge is harnessed. Even with modest data redundancy (7), a high-quality electron-density map was obtained from that single data set when combined with phase improvement methods. Two phasing protocols are compared for the same data set. Resolution extension (to 1.4 A), in this test, was also done but via a second data set. In future, with a tilted detector geometry, full diffraction resolution will be collectable in one experiment on the one beamline. Moreover, careful minimization of beamline Be window thickness on the new Daresbury SRS Multipole Wiggler Beamline 10 will maximize the 2-3 A (softer) wavelength intensity performance specification. The work has application to the harnessing of iodine f" optimized signals and enhanced sulfur f", as well as xenon f", in protein crystallography. This approach, softer-SWAT, should 'swat' many protein structures when it comes online.

Crystallography, X-Ray↗

PubMed2003

Low-resolution molecular replacement using a six-dimensional search.

A parallel-aware program MPI_FSEARCH has been implemented to perform molecular replacement using an exhaustive six-dimensional search. In particular, the program can be used to deal with diffraction data at very low resolution (d > 10 A) that would normally not be appropriate for other molecular-replacement programs such as AMoRe and CNS. Although an envelope constructed from a PDB (Protein Data Bank) file was tested in the present study, the program can be used to perform low-resolution molecular replacement with an envelope derived from various sources such as electron microscopy or small-angle solution X-ray scattering.

6-Phytase↗