Biomedical subjects

Zihe Rao

Publications and source records attributed to Zihe Rao.

At least 109 records · Page 6Linked to original sources

PubMed2002

Structure of the N-terminal domain of human FKBP52.

FKBP52 is a member of the FK506-binding protein family (FKBPs). The N-terminal domain of FKBP52 (FKBP52-N; residues 1-140) is responsible for peptidyl-prolyl isomerase activity and binding of FK506. Here, the crystal structure of FKBP52-N has been determined by molecular replacement to 2.4 A. FKBP52-N is defined by a six-stranded antiparallel beta-sheet wrapping with a right-handed twist around a short alpha-helix, an architecture similar to that of FKBP12. FKBP52-N is able to bind FK506 in a similar way to FKBP12. The variability in two loop regions (residues 70-76 and 108-127) is the principal reason for the specificity differences between FKBP52-N and FKBP12. The Pro120 change corresponding to Gly89 in FKBP12 limits the conformational adaptation between the loop (residues 108-127) and FK506 and decreases the FK506 affinity, while the Lys121 substitution corresponding to Ile90 of FKBP12 destroys a key interaction between FKBP52-N and calcineurin. It can be inferred from the locations of strictly conserved amino acids in the polypeptide chain that the maintenance of the overall conformation of the PPIase domains of FKBPs is essential for the PPIase activity. The N-terminal region and beta-sheets of FKBP52-N forms a hydrophobic patch which may be responsible for the binding of target proteins such as dynein or PAHX.

Amino Acid Sequence↗

PubMed2002

Crystallization and preliminary crystallographic analysis of a partial extracellular fragment of a sperm membrane protein YWK-II/APPH related to the Alzheimer betaA4-amyloid precursor protein.

Crystals of a partial extracellular fragment of a human sperm membrane protein YWK-II/APPH have been grown at 291 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.8 A resolution at 100 K using Cu K(alpha) radiation. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.009, b = 67.387, c = 149.241 A, alpha = beta = gamma = 90 degrees. The presence of two molecules per asymmetric unit gives a crystal Volume per protein mass (V(M)) of 3.51 A(3) Da(-1) and a solvent content of 64.6% by Volume. A full set of X-ray diffraction data were collected to 2.8 A resolution from the native crystal.

Amino Acid Sequence↗

PubMed2002

Crystallization and preliminary X-ray diffraction analysis of FKBP52 N-terminal domain.

FKBP52 is a FK506-binding protein which was first discovered in the heterocomplex composed of HSP90 and inactive steroid receptor. Here, the N-terminal domain (residues 1-140) of FKBP52 has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion technique. Crystals with a 2.4 A resolution limit were obtained using ammonium sulfate as precipitant at pH 8.5. The crystals belong to space group P2(1), with unit-cell parameters a = 27.8, b = 58.4, c = 70.9 A, beta = 98.3 degrees. Assuming two molecules per asymmetric unit, the solvent content is calculated to be 40%.

Crystallization↗

PubMed2002

Expression, crystallization and preliminary X-ray studies of the recombinant PTB domain of human dok-5 protein.

The human dok-5 PTB domain fusion protein has been overexpressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study. Crystals were obtained by the vapour-diffusion method. The crystal has unit-cell parameters a = b = 75.9, c = 108.0 A, alpha = beta = 90, gamma = 120 degrees and belongs to space group P3(2)21. Diffraction data were collected to 2.8 A resolution in-house. Furthermore, a selenomethionine (SeMet) derivative of dok-5 PTB domain fusion protein was overexpressed using the same expression system and was purified in a reductive environment. The derivative crystals were obtained under similar conditions. Subsequently, three different wavelength data sets were collected to 2.3 A resolution from the derivative crystal at the Advanced Photon Source, Argonne National Laboratory.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Crystal structure of the F87W/Y96F/V247L mutant of cytochrome P-450cam with 1,3,5-trichlorobenzene bound and further protein engineering for the oxidation of pentachlorobenzene and hexachlorobenzene.

We reported previously that the F87W/Y96F/V247L mutant of cytochrome P-450cam (CYP101) from Pseudomonas putida catalyzed the rapid oxidation of lightly chlorinated benzenes, but pentachlorobenzene oxidation was slow (Jones, J. P., O'Hare, E. J., and Wong, L. L. (2001) Eur. J. Biochem. 268, 1460-1467). In the present work, we determined the crystal structure of this mutant with bound 1,3,5-trichlorobenzene. The substrate was bound to crystallographically independent CYP101 molecules in at least three different orientations, which were distinguished by the angle between the benzene ring and the porphyrin, and one orientation contained an Fe-Cl interaction. In another orientation, the substrate was almost parallel to the heme, with a C-H bond closest to the iron. The enzyme/substrate contacts suggested that the L244A mutation should promote the binding of pentachlorobenzene and hexachlorobenzene by creating space to accommodate the extra chlorines. The F87W/Y96F/L244A/V247L mutant thus designed was found to oxidize pentachlorobenzene at a rate of 82.5 nmol (nmol CYP101)(-1) min(-1), 45 times faster than the F87W/Y96F/V247L parent mutant. The rate of hexachlorobenzene oxidation was increased 200-fold, to 2.0 min(-1). Both substrates are oxidized to pentachlorophenol, which is degraded by micro-organisms. In principle, the F87W/Y96F/L244A/V247L mutant could have applications in the bioremediation of polychlorinated benzenes.

Amino Acid Substitution↗

PubMed2002

Crystallographic study of a naturally occurring trans-splicing intein from Synechocystis sp. PCC 6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been purified and crystallized using PEG 8K as precipitant. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 58.5, c = 70.2 A. It has one molecule per asymmetric unit and diffracts to beyond 2.9 A under cryoconditions (110 K) using a Cu rotating-anode X-ray generator in-house.

Alternative Splicing↗

PubMed2002

Purification and initial crystallization studies of a DnaB intein from Synechocystis sp. PCC 6803.

A 154-residue mini-intein from the dnaB gene of Synechocystis sp. PCC 6803 (Ssp DnaB intein) has been purified and crystallized using PEG 4000 as a precipitant. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 58.2, c = 70.3 A. It has one molecule per asymmetric unit and diffracts to beyond 2.0 A under cryoconditions (100 K) using a rotating copper anode X-ray generator.

Bacterial Proteins↗

PubMed2002

Expression, purification, crystallization and preliminary X-ray analysis of a DNA-binding protein from Methanococcus jannaschii.

A small DNA-binding protein of 87 amino-acid residues from the hyperthermophilic archaeon Methanococcus jannaschii (Mja10b) was cloned and overexpressed in Escherichia coli. The protein was crystallized and the crystals belong to the space group P6(1)22/P6(5)22, with unit-cell parameters a = b = 50.85, c = 124.02 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffracted to a maximum resolution of 2.2 A at 100 K using Cu Kalpha radiation. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49% by volume. A full set of X-ray diffraction data was collected to 2.2 A from the native crystal.

Crystallography, X-Ray↗

PubMed2002

Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides.

Glycoprotein 96 (gp96), a member of heat shock protein (HSP) family, plays important roles in both innate immunity by itself and in T cell adaptive immunity in complex with antigenic-specific peptides. Using ammonium sulfate precipitation, ConA-Sepharose affinity chromatography and anion exchange chromatography, we have successfully purified gp96 from human liver tumor tissues. Subsequently, the gp96-associated peptides were successfully isolated from the gp96 preparation in quantities sufficient for micro-sequencing and identification of the peptides [Lancet 357 (2001) 528]. This three-step purification method might be used as a universal technique for the easy and reproducible isolation of antigenic heat shock protein gp96 from liver tissue or even other tissues of different sources. This will inevitably trigger the search for antigenic-specific peptides bound to gp96. Here, we report the method in detail.

Ammonium Sulfate↗

PubMed2002

Crystallization and preliminary crystallographic studies on the chromo shadow domain (CSD) of mouse heterochromatin protein M31.

Members of the heterochromatin protein 1 (HP1) class of non-histone chromosomal proteins are components of heterochromatin and are involved in the epigenetic regulation of the genome. HP1 proteins are modular and consist of two sequence-related domains called the chromodomain (CD) and the chromo shadow domain (CSD). In order to investigate the role of the murine HP1-like protein M31 in heterochromatin formation and gene silencing, recombinant CSD was overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method with PEG 4000 as precipitant. Diffraction data to 2.9 A were collected from a native crystal belonging to space group C222(1), with unit-cell parameters a = 60.0, b = 95.6, c = 91.7 A, alpha = beta = gamma = 90 degrees.

Animals↗

PubMed2002

Crystallization and preliminary crystallographic analysis of acylamino-acid releasing enzyme from the hyperthermophilic archaeon Aeropyrum pernix.

Crystals of acylamino-acid releasing enzyme from the hyperthermophilic archaeon Aeropyrum pernix strain K1 have been grown at 291 K using ammonium phosphate as a precipitant. The diffraction pattern of the crystal extends to 2.4 A resolution at 100 K using Cu Kalpha radiation. The crystal belongs to space group P1, with unit-cell parameters a = 107.5, b = 109.9, c = 119.4 A, alpha = 108.1, beta = 109.8, gamma = 91.9 degrees. The presence of eight molecules per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 48% by volume. A full set of X-ray diffraction data was collected to 2.9 A from the native crystal.

Archaeal Proteins↗

PubMed2002

Crystallization and preliminary X-ray analysis of recombinant histone HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Recombinant archaeal histone from the hyperthermophile Pyrococcus horikoshii OT3 (HPhA) was crystallized by the hanging-drop vapour-diffusion method. Crystals grew at 291 K in 200 mM (NH(4))(2)SO(4), 100 mM sodium acetate buffer pH 4.6, 19% PEG 4000. Diffraction data were obtained to a resolution of 2.3 A from a single frozen crystal, which belonged to space group P2(1) with unit-cell parameters a = 34.99, b = 46.89, c = 35.02 A, alpha = gamma = 90, beta = 104 degrees. The asymmetric unit contained two molecules and had a solvent content of approximately 35%.

Crystallization↗

PubMed2002

Purification, crystallization and preliminary X-ray diffraction studies of a Ca2+-binding protein, human S100P.

S100P, a Ca(2+)-binding protein, is a member of the S100 family. Its presence is associated with the development of prostate cancer, but its cellular function is not known. Recombinant human S100P has been expressed and purified in bacterial cells and crystals of human S100P in the calcium-bound state have been grown using the vapour-diffusion technique with PEG 4000 as precipitant. Diffraction data have been obtained to a resolution of 2.0 A from a single frozen S100P crystal which belongs to the space group P4(1)2(1)2, with unit-cell parameters a = b = 60.8, c = 47.6 A.

Calcium-Binding Proteins↗

PubMed2002

Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.

Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.

Amidohydrolases↗

PubMed2002

Atomic resolution structure of Moloney murine leukemia virus matrix protein and its relationship to other retroviral matrix proteins.

Matrix proteins associated with the viral membrane are important in the formation of the viral particle and in virus maturation. The 1.0 A crystal structure of the ecotropic Gammaretrovirus Moloney murine leukemia virus (M-MuLV) matrix protein reveals the conserved topology of other retroviral matrix proteins, despite undetectable sequence similarity. The N terminus (normally myristylated) is exposed and adjacent to a basic surface patch, features likely to contribute to membrane binding. The four proteins in the asymmetric unit make varied contacts. The M-MuLV matrix structure is intermediate, between those of the lentiviruses and other retroviruses. The protein fold appears to be maintained, in part, by the conservation of side chain packing, which may provide a useful tool for searching for weak distant similarities in proteins.

Amino Acid Sequence↗

PubMed2002

Molecular coordination of alphabeta T-cell receptors and coreceptors CD8 and CD4 in their recognition of peptide-MHC ligands.

The interaction of the alphabeta T-cell receptor (TCR) with its ligand, peptide-MHC (pMHC), is enhanced by the recognition of the coreceptor CD8 or CD4 to the same pMHC in the immunological synapse. In the past few years, the coordination of these interactions at the molecular level has been revealed by analysis of their complex crystal structures and binding dynamics. Here we discuss the interactions of pMHC with the TCR and coreceptor CD8 or CD4 on the surfaces of alphabeta T cells and antigen presenting cells, and the implications for TCR signalling and the T-cell repertoire.

Animals↗

PubMed2002

Crystal structure of a staphylokinase: variant a model for reduced antigenicity.

Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-A resolution, could explain a major antigenic epitope (residues A72-F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK.

Antigens, Bacterial↗

PubMed2002

Crystal structure of the catalytic domain of a human thioredoxin-like protein.

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.

Catalytic Domain↗