Biomedical subjects

Yiwei Liu

Publications and source records attributed to Yiwei Liu.

31 records · Page 2Linked to original sources

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 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↗

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

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

The crystal structure of a TL/CD8alphaalpha complex at 2.1 A resolution: implications for modulation of T cell activation and memory.

TL is a nonclassical MHC class I molecule that modulates T cell activation through relatively high-affinity interaction with CD8alphaalpha. To investigate how the TL/CD8alphaalpha interaction influences TCR signaling, we characterized the structure of the TL/CD8alphaalpha complex using X-ray crystallography. Unlike antigen-presenting molecules, the TL antigen-binding groove is occluded by specific conformational changes. This feature eliminates antigen presentation, severely hampers direct TCR recognition, and prevents TL from participating in the TCR activation complex. At the same time, the TL/CD8alphaalpha interaction is strengthened through subtle structure changes in the TL alpha3 domain. Thus, TL functions to sequester and redirect CD8alphaalpha away from the TCR, modifying lck-dependent signaling.

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↗

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

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↗

PubMed2001

Crystallization and preliminary crystallographic analysis of a metastasis-inducing protein, human S100A4.

A metastasis-inducing protein, human S100A4, cDNA was overexpressed in Escherichia coli. The recombinant protein was purified by three chromatographic procedures. Crystals were obtained using polyethylene glycol as a precipitant. The native S100A4 crystals did not diffract sufficiently well for data collection, but the SeMet S100A4 crystal, grown using similar conditions as for the native, was found to be stable during exposure to X-rays and diffracted to 4 A resolution in-house. The crystal belongs to space group P6 or P3. The unit-cell parameters are a = b = 47.1, c = 175.6 A, alpha = beta = 90, gamma = 120 degrees. Complete data will be collected using synchrotron radiation.

Crystallography, X-Ray↗