Biomedical subjects

Zihe Rao

Publications and source records attributed to Zihe Rao.

At least 91 records · Page 5Linked to original sources

PubMed2003

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.

Animals↗

PubMed2003

Crystal structure of alanine:glyoxylate aminotransferase and the relationship between genotype and enzymatic phenotype in primary hyperoxaluria type 1.

A deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is responsible for the potentially lethal hereditary kidney stone disease primary hyperoxaluria type 1 (PH1). Many of the mutations in the gene encoding AGT are associated with specific enzymatic phenotypes such as accelerated proteolysis (Ser205Pro), intra-peroxisomal aggregation (Gly41Arg), inhibition of pyridoxal phosphate binding and loss of catalytic activity (Gly82Glu), and peroxisome-to-mitochondrion mistargeting (Gly170Arg). Several mutations, including that responsible for AGT mistargeting, co-segregate and interact synergistically with a Pro11Leu polymorphism found at high frequency in the normal population. In order to gain further insights into the mechanistic link between genotype and enzymatic phenotype in PH1, we have determined the crystal structure of normal human AGT complexed to the competitive inhibitor amino-oxyacetic acid to 2.5A. Analysis of this structure allows the effects of these mutations and polymorphism to be rationalised in terms of AGT tertiary and quaternary conformation, and in particular it provides a possible explanation for the Pro11Leu-Gly170Arg synergism that leads to AGT mistargeting.

Alleles↗

PubMed2003

Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing.

We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.

Amino Acid Sequence↗

PubMed2003

Crystallization and preliminary X-ray diffraction analysis of post-fusion six-helix bundle core structure from Newcastle disease virus F protein.

Fusion of virus members from the Paramyxoviridae family involves two glycoproteins. They are termed attachment glycoprotein (HN, H or G) and fusion protein (F). The F protein contains two highly conserved heptad-repeat (HR) regions, HR1 and HR2. Through conformational changes in the F protein, HR1 and HR2 are believed to form a stable six-helix coiled-coil bundle during the membrane-fusion process. However, no crystal structure has yet been documented for this state in the Newcastle disease virus (NDV, a member of the Paramyxoviridae family) F protein, despite the recent success on its F(0) crystal structure (Chen et al., 2001), which was thought to represent the pre-fusion conformation of F glycoprotein. In this study, a single-chain polypeptide constructed by linking two truncated HR regions of the NDV F protein has been expressed, purified and crystallized by means of the hanging- or sitting-drop vapour-diffusion method. Crystals in hexagonal and trapezoid forms with a resolution limit of 2.6 A were obtained. These crystals belonged to space group C2, with unit-cell parameters a = 66.4, b = 38.2, c = 102.0 A, beta = 100.2 degrees. Crystals in the rhombic form with a resolution limit of 2.5 A were also obtained. These crystals belonged to space group P2(1), with unit-cell parameters a = 59.0, b = 31.9, c = 62.3 A, beta = 117.0 degrees. This will add to the repertoire of viral fusion protein post-fusion state structures and help further the understanding of the molecular mechanism of enveloped virus fusion.

Crystallization↗

PubMed2003

Crystal structure of the ectodomain of human FcalphaRI.

Human FcalphaRI (CD89) is the receptor specific for IgA, an immunoglobulin that is abundant in mucosa and is also found in high concentrations in serum. Although FcalphaRI is an immunoglobulin Fc receptor (FcR), it differs in many ways from FcRs for other immunoglobulin classes. The genes of most FcRs are located on chromosome 1 at 1q21-23, whereas FcalphaRI is on chromosome 19, at 19q13.4, a region called the leukocyte receptor complex, because it is clustered with several leukocyte receptor families including killer cell inhibitory receptors (KIRs) and leukocyte Ig-like receptors (LIRs). The amino acid sequence of FcalphaRI shares only 20% homology with other FcRs but it has around 35% homology with its neighboring LIRs and KIRs. In this work, we analyzed the crystal structure of the ectodomain of FcalphaRI and examined structure similarities between FcalphaRI and KIR2DL1, KIR2DL2 and LIR-1. Our data show that FcalphaRI, KIRs, and LIRs share a common hydrophobic core in their interdomain interface, and FcalphaRI is evolutionally closer to LIR than KIR.

Amino Acid Sequence↗

PubMed2003

Crystallization and preliminary crystallographic analysis of extracellular fragment X3 of YWK-II/APPH: a human sperm membrane protein related to the Alzheimer betaA4-amyloid precursor protein.

Crystals of extracellular fragment X3 of a human sperm membrane protein YWK-II/APPH have been grown at 291 K using 8% PEG 4000 as precipitant by the vapour-diffusion method. The diffraction pattern of the crystal extends to 2.9 A resolution at 100 K using Cu Kalpha radiation in-house. The crystals belong to space group P2(1), with unit-cell parameters a = 46.0, b = 43.7, c = 90.2 A, alpha = gamma = 90.0, beta = 106.6 degrees. Furthermore, a selenomethionine (SeMet) derivative of the protein was overexpressed in the same expression system and was purified in a reducing environment. The derivative crystals were obtained under similar conditions. Subsequently, a single-wavelength data set was collected to 2.38 A resolution from the derivative crystal at ESRF. The crystals belong to space group P2(1), with unit-cell parameters a = 46.2, b = 44.0, c = 88.3 A, alpha = gamma = 90.0, beta = 103.6 degrees. The presence of one molecule per asymmetric unit gives a crystal Volume per protein mass (V(M)) of 2.8 A(3) Da(-1) and a solvent content of 56.4% by Volume.

Amyloid beta-Protein Precursor↗

PubMed2003

Crystallization and preliminary X-ray crystallographic analysis of the trimer core from measles virus fusion protein.

Two heptad-repeat regions (HR1 and HR2) are highly conserved in paramyxovirus fusion proteins and form a stable helical trimer of heterodimers [(HR1-HR2)(3)] after the fusion between viral and cellular membranes. In this study, two HR regions of the fusion protein of measles virus, a member of the paramyxoviruses, were selected and overexpressed as a single chain (named 2-Helix) connected by an amino-acid linker using a GST-fusion expression system in Escherichia coli. Crystals of 2-Helix protein (GST removed) could be obtained from many conditions using the sitting- or hanging-drop vapour-diffusion method. A complete data set was collected in-house to 1.9 A resolution from a single crystal. The crystal belongs to space group P6, with unit-cell parameters a = b = 51.637, c = 67.058 A. To facilitate the crystal structure solution, SeMet-substituted 2-Helix crystals, grown under similar conditions to the native, were also obtained and diffracted X-rays to 1.8 A using synchrotron radiation.

Crystallization↗

PubMed2003

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation.

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

Acetyl Coenzyme A↗

PubMed2003

Structure based hyperthermostability of archaeal histone HPhA from Pyrococcus horikoshii.

The histone protein HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii, shows hyperthermostability, as required for optimal growth of the organism at 95 degrees C. The structure of recombinant P.horikoshii HPhA has been determined to 2.3A resolution by molecular replacement, and refined to R(work) and R(free) values of 20.7% and 27.3%, respectively. The HPhA monomer structure is characterized by the histone fold and assembles into a homodimer like other archaeal histones. There are four anions found in the dimer structure, giving rise to clues as to where DNA might bind. A detailed comparison of four known structures of archaeal histones, which evolve and exist under different temperatures, shows that the thermophilic archaeal histone HPhA has a larger hydrophobic contact area, an increased number of hydrogen bonds and a reduced solvent-accessible area. We also observe a unique network of tyrosine residues located at the crossover point of the two HPhA monomers, which locks them together and stabilizes the dimer. These factors together account for the increased thermal stability.

Amino Acid Sequence↗

PubMed2003

The crystal structure at 2A resolution of the Ca2+ -binding protein S100P.

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.

Amino Acid Sequence↗

PubMed2003

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam.

Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam). Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis. (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.

Bicyclic Monoterpenes↗

PubMed2003

Design and structure-based study of new potential FKBP12 inhibitors.

Based on the structure of FKBP12 complexed with FK506 or rapamycin, with computer-aided design, two neurotrophic ligands, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester and (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-phenylalanine benzyl ester, were designed and synthesized. Fluorescence experiments were used to detect the binding affinity between FKBP12 and these two ligands. Complex structures of FKBP12 with these two ligands were obtained by x-ray crystallography. In comparing FKBP12-rapamycin complex and FKBP12-FK506 complex as well as FKBP12-GPI-1046 solution structure with these new complexes, significant volume and surface area effects and obvious contact changes were detected which are expected to cause their different binding energies-showing these two novel ligands will become more effective neuron regeneration drugs than GPI-1046, which is currently undergoing phase II clinical trail as a neurotrophic drug. Analysis of volume and surface area effects also gives a new clue for structure-based drug design.

Binding Sites↗

PubMed2003

Design and analysis of post-fusion 6-helix bundle of heptad repeat regions from Newcastle disease virus F protein.

Fusion of paramyxovirus to the cell involves receptor binding of the HN glycoprotein and a number of conformational changes of F glycoprotein. The F protein is expressed as a homotrimer on the virus surface. In the present model, there are at least three conformations of F protein, i.e. native form, pre-hairpin intermediate and the post-fusion state. In the post-fusion state, the two highly conserved heptad repeat (HR) regions of F protein form a stable 6-helix coiled-coil bundle. However, no crystal structure is known for this state for the Newcastle disease virus, although the crystal structure of the F protein native form has been solved recently. Here we deployed an Escherichia coli in vitro expression system to engineer this 6-helix bundle by fusion of either the two HR regions (HR1, linker and HR2) or linking the 6-helix [3 x (HR1, linker and HR2)] together as a single chain. Subsequently, both of them form a stable 6-helix bundle in vitro judging by gel filtration and chemical cross-linking and the proteins show salient features of an alpha-helix structure. Crystals diffracting X-rays have been obtained from both protein preparations and the structure determination is under way. This method could be used for crystallization of the post-fusion state HR structures of other viruses.

Amino Acid Sequence↗

PubMed2003

The structural basis of Trp192 and the C-terminal region in trichosanthin for activity and conformational stability.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing N-glycosidase activity. TCS has various pharmacological properties, including immunomodulatory, anti-tumor and anti-HIV activities. Up to seven C-terminal residues of TCS (TCS-C7) can be deleted resulting in lower antigenicity with minimal effects on its activity. However, an additional problem is that the minimal effects on activity are higher than the reduction in antigenicity. In the present work, the crystal structure of TCS-C7 was determined. It shows the details of the C-terminal residues of TCS-C7, and in particular the hydrogen bonds between P35 and L240, S196 and L240, and W192 and L239, which play an important role in maintaining the structure of TCS-C7. Further analysis shows that the hydrogen bonds related to Leu240 are key in maintaining the relationship between N- and C-terminal domains. The major role of the C-terminal tail appears to stabilize the structure of TCS. The conformation between helix H7 at the N-terminal domain and the C-terminal tail at the C-terminal domain is also revealed. Two mutants, TCS-W192F and TCS-C7-W192F, were prepared and crystal structures were determined. These variants have greatly reduced ribosome-inactivating activities compared with TCS and TCS-C7, respectively, and TCS-W192F and TCS-C7-W192F have a similar stability in guanidine hydrochloride compared with TCS-C7. This suggests that Trp192 can affect the ribosome-inactivating activity of TCS.

Crystallography, X-Ray↗

PubMed2003

Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have been revealed. Here we report the crystal structures of several E85 mutant TCS complexes with adenosine-5'-monophosphate (AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near the active site of the molecule and parallel to the aromatic ring of Tyr70, an AMP molecule is bound to the mutant without being hydrolyzed. In the E85R TCS/adenine complex, the hydrolyzed product adenine is located in the active pocket where it occupies a position similar to that in the TCS/NADPH complex. Significantly, AMP is bound in a position different to that of adenine. In comparison with these structures, we suggest that there are at least two subsites in the active site of TCS, one for initial substrate recognition as revealed by the AMP site and another for catalysis as represented by the NADPH site. Based on these complex structures, the function of residue 85 and the mechanism of catalysis are proposed.

Adenine↗

PubMed2003

Crystal structure of a DNA binding protein from the hyperthermophilic euryarchaeon Methanococcus jannaschii.

The Sac10b family consists of a group of highly conserved DNA binding proteins from both the euryarchaeotal and the crenarchaeotal branches of Archaea. The proteins have been suggested to play an architectural role in the chromosomal organization in these organisms. Previous studies have mainly focused on the Sac10b proteins from the crenarchaeota. Here, we report the 2.0 A resolution crystal structure of Mja10b from the euryarchaeon Methanococcus jannaschii. The model of Mja10b has been refined to an R-factor of 20.9%. The crystal structure of an Mja10b monomer reveals an alpha/beta structure of four beta-strands and two alpha-helices, and Mja10b assembles into a dimer via an extensive hydrophobic interface. Mja10b has a similar topology to that of its crenarchaeota counterpart Sso10b (also known as Alba). Structural comparison between the two proteins suggests that structural features such as hydrophobic inner core, acetylation sites, dimer interface, and DNA binding surface are conserved among Sac10b proteins. Structural differences between the two proteins were found in the loops. To understand the structural basis for the thermostability of Mja10b, the Mja10b structure was compared to other proteins with similar topology. Our data suggest that extensive ion-pair networks, optimized accessible surface area and the dimerization via hydrophobic interactions may contribute to the enhanced thermostability of Mja10b.

Amino Acid Motifs↗

PubMed2003

Crystal structure of Swine vesicular disease virus and implications for host adaptation.

Swine vesicular disease virus (SVDV) is an Enterovirus of the family Picornaviridae that causes symptoms indistinguishable from those of foot-and-mouth disease virus. Phylogenetic studies suggest that it is a recently evolved genetic sublineage of the important human pathogen coxsackievirus B5 (CBV5), and in agreement with this, it has been shown to utilize the coxsackie and adenovirus receptor (CAR) for cell entry. The 3.0-A crystal structure of strain UK/27/72 SVDV (highly virulent) reveals the expected similarity in core structure to those of other picornaviruses, showing most similarity to the closest available structure to CBV5, that of coxsackievirus B3 (CBV3). Features that help to cement together and rigidify the protein subunits are extended in this virus, perhaps explaining its extreme tolerance of environmental factors. Using the large number of capsid sequences available for both SVDV and CBV5, we have mapped the amino acid substitutions that may have occurred during the supposed adaptation of SVDV to a new host onto the structure of SVDV and a model of the SVDV/CAR complex generated by reference to the cryo-electron microscopy-visualized complex of CBV3 and CAR. The changes fall into three clusters as follows: one lines the fivefold pore, a second maps to the CAR-binding site and partially overlaps the site for decay accelerating factor (DAF) to bind to echovirus 7 (ECHO7), and the third lies close to the fivefold axis, where the low-density lipoprotein receptor binds to the minor group of rhinoviruses. Later changes in SVDV (post-1971) map to the first two clusters and may, by optimizing recognition of a pig CAR and/or DAF homologue, have improved the adaptation of the virus to pigs.

Adaptation, Physiological↗

PubMed2003

Site-directed mutagenesis and preliminary x-ray crystallographic studies of the tabtoxin resistance protein.

Tabtoxin resistance protein (TTR) is an enzyme that catalyzes the acetylation of tabtoxin rendering tabtoxin-producing pathogens tolerant to their own phytotoxins. According to the structure based detoxification mechanism of TTR, three site-directed mutants Y141F, D130N and Y141F-D130N were constructed and overexpressed in E. coli. The products were then purified and their properties were analyzed by CD and DLS. Crystallization trials of two mutants Y141F andY141F-D130N were preformed.

Acetylation↗