Biomedical subjects

Zihe Rao

Publications and source records attributed to Zihe Rao.

At least 73 records · Page 4Linked to original sources

PubMed2004

3D structure of human FK506-binding protein 52: implications for the assembly of the glucocorticoid receptor/Hsp90/immunophilin heterocomplex.

FK506-binding protein 52 (FKBP52), which binds FK506 and possesses peptidylprolyl isomerase activity, is an important immunophilin involved in the heterocomplex of steroid receptors with heat-shock protein 90. Here we report the crystal structures of two overlapped fragments [N(1-260) and C(145-459)] of FKBP52 and the complex with a C-terminal pentapeptide from heat-shock protein 90. Based on the structures of these two overlapped fragments, the complete putative structure of FKBP52 can be defined. The structure of FKBP52 is composed of two consecutive FKBP domains, a tetratricopeptide repeat domain and a short helical domain beyond the final tetratricopeptide repeat motif. Key structural differences between FKBP52 and FKBP51, including the relative orientations of the four domains and some important residue substitutions, could account for the differential functions of FKBPs.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary crystallographic analysis of the fusion core from two new zoonotic paramyxoviruses, Nipah virus and Hendra virus.

Highly conserved heptad-repeat (HR1 and HR2) regions in class I viral fusion (F) proteins, including the F protein from paramyxovirus, interact with each other post-fusion to form a six-helix bundle called a fusion core. Crystals of the fusion core of Nipah virus have been grown at 291 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.1 angstroms resolution at 100 K in-house. The crystals have unit-cell parameters a = 31.664, b = 31.725, c = 51.256 angstroms, alpha = 80.706, beta = 86.343, gamma = 65.812 degrees and belong to space group P1. Crystals of the fusion core of Hendra virus have also been grown at 291 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.0 angstroms resolution at 100 K in-house. A selenomethionine (SeMet) derivative of the HeV fusion core was overexpressed using the same Escherichia coli expression system and purified. The derivative crystals were obtained under similar conditions and three different wavelength data sets were collected to 2.0 angstroms resolution from the derivative crystal at BSRF (Beijing Synchrotron Radiation Facility). The crystals have unit-cell parameters a = 31.997, b = 31.970, c = 53.865 angstroms, alpha = 85.990, beta = 85.842, gamma = 68.245 degrees and belong to space group P1.

Crystallization↗

PubMed2004

Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core.

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.

Amino Acid Motifs↗

PubMed2004

Crystallization and preliminary crystallographic analysis of a native chitinase from the fungal pathogen Aspergillus fumigatus YJ-407.

Chitinase hydrolyzes chitin, a linear polymer of beta-1,4-linked N-acetylglucosamine (NAG), and plays a variety of roles in the biological world. In addition to endo- and exo-hydrolytic activities, transglycosyl activity has also been observed in the extracellular chitinase (afCHI) from the airborne saprophytic fungi Aspergillus fumigatus YJ-407. Crystals of this native chitinase have been grown at 291 K using PEG 3350 as a precipitant. The diffraction data from the crystal extend to 1.7 A resolution at BSRF, China. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 95.7, b = 100.5, c = 134.3 A. The presence of two molecules per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.6 A(3) Da(-1) and a solvent content of 65% by volume. A full set of X-ray diffraction data was collected to 2.1 A resolution.

Aspergillus fumigatus↗

PubMed2004

Crystallization and preliminary X-ray studies of methyl parathion hydrolase from Pseudomonas sp. WBC-3.

Methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3, an enzyme that catalyzes the degradation of methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate; MP), has been purified and crystallized by the hanging-drop vapour-diffusion method. The crystals were grown at 291 K using a precipitant solution consisting of 30% PEG 400, 0.1 M sodium acetate pH 4.6, 0.1 M CdCl(2). MPH is a zinc-containing enzyme judged by inductively coupled plasma mass-spectrometric (ICP-MS) analysis. Multiple-wavelength anomalous dispersive X-ray data were collected at 2.5 A resolution from a single crystal on beamline 41XU at SPring-8. The crystal belongs to space group P4(3)2(1)2, with unit-cell parameters a = 84.94, b = 84.94, c = 200.38 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two molecules and has a solvent content of approximately 52%. Crystal structure determination is in progress.

Crystallization↗

PubMed2004

Basis for fusion inhibition by peptides: analysis of the heptad repeat regions of the fusion proteins from Nipah and Hendra viruses, newly emergent zoonotic paramyxoviruses.

Nipah virus (NiV) and Hendra virus (HeV) are novel zoonotic members of the Paramyxoviridae family and are the prototypes for a newly designated genus, Genus Henipavirus. Recent studies have shown that paramyxovirus might adopt a similar mechanism of virus fusion-entry. Under this mechanism, the two highly conserved heptad repeat (HR) regions, HR1 and HR2, in the fusion (F) protein, seem to show characteristic structure in the fusion core: the formation of a 6-helix coiled-coil bundle. The three HR1s form the alpha-helix coiled-coil surrounded by three HR2s. In this study, the two HR regions of NiV or HeV were expressed in an Escherichia coli system as a single chain and the results do show that HR1 and HR2 interact with each other in both NiV and HeV and form typical 6-helix coiled-coil bundles. This provides the molecular basis of HR2 inhibition to NiV and HeV fusion as observed in an earlier report.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary X-ray analysis of inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Inorganic pyrophosphatase (PPase; EC 3.6.1.1) from the hyperthermophile Pyrococcus horikoshii was crystallized by the hanging-drop vapour-diffusion method at pH 5.0 using polyethyleneglycol 4000 as the precipitant. The crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 71.7, b = 86.5, c = 92.5 A, alpha = beta = gamma = 90 degrees. There are two molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and a full set of X-ray diffraction data was collected to 2.7 A resolution in-house.

Crystallization↗

PubMed2004

Expression, crystallization and preliminary X-ray studies of the recombinant PTB domain of mouse dok1 protein.

The PTB domain of mouse dok1 fusion protein has been overexpressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study. Crystals have been obtained using the vapour-diffusion method and belong to space group P2(1)2(1)2(1). X-ray diffraction data were collected in-house to 2.5 A resolution. A selenomethionine (SeMet) dok1 PTB fusion-protein derivative was expressed using the same expression system, purified in a reductive environment and crystals were obtained under similar conditions. Subsequently, three different wavelength data sets from the derivative crystal were collected to 2.5 A resolution at SPring-8.

Animals↗

PubMed2004

Construct design, biophysical, and biochemical characterization of the fusion core from mouse hepatitis virus (a coronavirus) spike protein.

Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the formation of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been confirmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a flexible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bundle has been analyzed by a series of biophysical and biochemical techniques and confirmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses.

Amino Acid Sequence↗

PubMed2004

Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1.

Acylpeptide hydrolases (APH; also known as acylamino acid releasing enzyme) catalyze the removal of an N-acylated amino acid from blocked peptides. The crystal structure of an APH from the thermophilic archaeon Aeropyrum pernix K1 to 2.1 A resolution confirms it to be a member of the prolyl oligopeptidase family of serine proteases. The structure of apAPH is a symmetric homodimer with each subunit comprised of two domains. The N-terminal domain is a regular seven-bladed beta-propeller, while the C-terminal domain has a canonical alpha/beta hydrolase fold and includes the active site and a conserved Ser445-Asp524-His556 catalytic triad. The complex structure of apAPH with an organophosphorus substrate, p-nitrophenyl phosphate, has also been determined. The complex structure unambiguously maps out the substrate binding pocket and provides a basis for substrate recognition by apAPH. A conserved mechanism for protein degradation from archaea to mammals is suggested by the structural features of apAPH.

Aeropyrum↗

PubMed2004

Crystal structure of the hyperthermophilic inorganic pyrophosphatase from the archaeon Pyrococcus horikoshii.

A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88 degrees C. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 A resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia coli shows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.

Amino Acid Sequence↗

PubMed2004

Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine.

Some of the structural proteins of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) carry major epitopes involved in virus neutralization and are essential for the induction of protective humoral responses and the development of an effective vaccine. Rabbit antisera were prepared using full-length N and M proteins and eight expressed fragments covering the S protein. Antisera to S and M proteins were found to have different neutralizing titres towards SARS-CoV infection in vivo, ranging from 1:35 to 1:128. Antiserum to the N protein did not contain neutralizing antibodies. Epitopes inducing protective humoral responses to virus infection were located mainly in the M protein and a region spanning residues 13-877 of the S protein. The neutralizing ability of antisera directed against the expressed structural proteins was greater than that of convalescent patient antisera, confirming that, as immunogens, the former induce strong, SARS-CoV-specific neutralizing antibody responses. The in vitro neutralization assay has important implications for the design of an effective, protein-based vaccine preventing SARS-CoV infection.

Animals↗

PubMed2003

Crystallization and preliminary crystallographic studies of the C-terminal domain of human FKBP52.

FKBP52 is a high-molecular-weight immunophilin belonging to the FKBP (FK506-binding protein) family. FKBP52 is one of several chaperone proteins associated with untransformed steroid receptors in steroid receptor-hsp90 heterocomplexes. Here, the C-terminal domain (amino acids 145-459) has been cloned, overexpressed and purified. Crystals were obtained using the hanging-drop vapour-diffusion technique with ammonium sulfate as precipitant in 0.1 M Tris pH 8.0 solution. Diffraction data to 2.7 A were collected from a selenomethionine-containing crystal belonging to space group C222(1), with unit-cell parameters a = 114.4, b = 143.1, c = 171.2 A, alpha = beta = gamma = 90 degrees. There are three molecules per asymmetric unit.

Amino Acid Sequence↗

PubMed2003

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↗

PubMed2003

Crystallization and preliminary crystallographic analysis of the extracellular fragment of FcalphaRI/CD89.

Crystals of the extracellular fragment of FcalphaRI/CD89 have been grown at 291 K using PEG 8000 as precipitant. The diffraction pattern of the selenomethionine (SeMet) derivative crystal extended to 2.1 A resolution at SPring-8, Japan. The crystals belong to space group C222(1), with unit-cell parameters a = 59.0, b = 69.4, c = 106.3 A, alpha = beta = gamma = 90 degrees. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.12 A(3) Da(-1) and a solvent content of 60.2% by volume. A full set of X-ray diffraction data were collected to 2.1 A from an SeMet-derivative crystal.

Antigens, CD↗

PubMed2003

Structural basis for the specific recognition of RET by the Dok1 phosphotyrosine binding domain.

Dok1 is a common substrate of activated protein-tyrosine kinases. It is rapidly tyrosine-phosphorylated in response to receptor tyrosine activation and interacts with ras GTPase-activating protein and Nck, leading to inhibition of ras signaling pathway activation and the c-Jun N-terminal kinase (JNK) and c-Jun activation, respectively. In chronic myelogenous leukemia cells, it has shown constitutive phosphorylation. The N-terminal phosphotyrosine binding (PTB) domain of Dok1 can recognize and bind specifically to phosphotyrosine-containing motifs of receptors. Here we report the crystal structure of the Dok1 PTB domain alone and in complex with a phosphopeptide derived from RET receptor tyrosine kinase. The structure consists of a beta-sandwich composed of two nearly orthogonal, 7-stranded, antiparallel beta-sheets, and it is capped at one side by a C-terminal alpha-helix. The RET phosphopeptide binds to Dok1 via a surface groove formed between strand beta5 and the C-terminal alpha-helix of the PTB domain. The structures reveal the molecular basis for the specific recognition of RET by the Dok1 PTB domain. We also show that Dok1 does not recognize peptide sequences from TrkA and IL-4, which are recognized by Shc and IRS1, respectively.

Amino Acid Sequence↗

PubMed2003

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.

Amino Acid Chloromethyl Ketones↗

PubMed2003

Crystal structure of human pirin: an iron-binding nuclear protein and transcription cofactor.

Pirin is a newly identified nuclear protein that interacts with the oncoprotein B-cell lymphoma 3-encoded (Bcl-3) and nuclear factor I (NFI). The crystal structure of human Pirin at 2.1-A resolution shows it to be a member of the functionally diverse cupin superfamily. The structure comprises two beta-barrel domains, with an Fe(II) cofactor bound within the cavity of the N-terminal domain. These findings suggest an enzymatic role for Pirin, most likely in biological redox reactions involving oxygen, and provide compelling evidence that Pirin requires the participation of the metal ion for its interaction with Bcl-3 to co-regulate the NF-kappaB transcription pathway and the interaction with NFI in DNA replication. Substitution of iron by heavy metals thus provides a novel pathway for these metals to directly influence gene transcription. The structure suggests an interesting new role of iron in biology and that Pirin may be involved in novel mechanisms of gene regulation.

Amino Acid Sequence↗