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Wah Chiu

Publications and source records attributed to Wah Chiu.

16 recordsLinked to original sources
PubMed2003

Architecture of the herpes simplex virus major capsid protein derived from structural bioinformatics.

The dispositions of 39 alpha helices of greater than 2.5 turns and four beta sheets in the major capsid protein (VP5, 149 kDa) of herpes simplex virus type 1 were identified by computational and visualization analysis from the 8.5A electron cryomicroscopy structure of the whole capsid. The assignment of helices in the VP5 upper domain was validated by comparison with the recently determined crystal structure of this region. Analysis of the spatial arrangement of helices in the middle domain of VP5 revealed that the organization of a tightly associated bundle of ten helices closely resembled that of a domain fold found in the annexin family of proteins. Structure-based sequence searches suggested that sequences in both the N and C-terminal portions of the VP5 sequence contribute to this domain. The long helices seen in the floor domain of VP5 form an interconnected network within and across capsomeres. The combined structural and sequence-based informatics has led to an architectural model of VP5. This model placed in the context of the capsid provides insights into the strategies used to achieve viral capsid stability.

Amino Acid Motifs↗
PubMed2003

Structure of the type 1 inositol 1,4,5-trisphosphate receptor revealed by electron cryomicroscopy.

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.

Animals↗
PubMed2003

Structure of the herpesvirus major capsid protein.

Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus. We report the 2.9 A crystal structure of this fragment and its modeling into an 8.5 A resolution electron cryomicroscopy map of the HSV-1 capsid. The structure, the first for any capsid protein from Herpesviridae, revealed a novel fold, placing herpesviruses outside any of the structurally linked viral groupings. Alterations in the geometrical arrangements of the VP5 subunits in the capsomeres exposes different residues, resulting in the differential association of the tegument and VP26 with the pentons and hexons, respectively. The rearrangements of VP5 subunits required to form both pentavalent and hexavalent capsomeres result in structures that exhibit very different electrostatic properties. These differences may mediate the binding and release of other structural proteins during capsid maturation.

Capsid Proteins↗
PubMed2003

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP.

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.

Animals↗
PubMed2003

Studying large viruses.

In this article we have attempted to describe some structural aspects of large viruses. Although this may seem a straightforward task, it is complicated by the fact that large viruses do not represent a distinctive class of organisms and any grouping under this heading will include a range of unrelated viruses with different structures, replication strategies, and host types. To simplify matters we limited our definition to dsDNA viruses with genomes of 100 kbp or larger. However, even this restricted grouping includes viruses with diverse and seemingly unrelated structures. Furthermore, few if any structural features are exclusive to large viruses and most of what appears distinctive about their structure or assembly can also be found in smaller, and usually better characterized, viruses. Therefore we have not attempted to provide a comprehensive catalog of the properties of large viruses but have tried to illustrate particular structural points with examples from a few of the better known forms, notably herpes simplex virus (HSV) and phage T4. The two techniques used to provide rigorous analyses of virus structures are X-ray crystallography and electron cryomicroscopy with computer-assisted reconstruction. To date, X-ray crystallography has been successful only with smaller viruses, and what is known about the structures of these large viruses has come primarily from electron cryomicroscopy. However, with the notable exception of the HSV capsid, such studies have been limited in extent and of relatively low resolution, and the information obtained has been confined largely to describing the spatial distributions and relationships between the subunits. Nevertheless, these studies have given us our clearest insights into the biology of these complex particles and increases in resolution promise to extend these insights by bridging the gap between gross and atomic structures, as exemplified by the identification and mapping of secondary structural elements in the HSV capsid.

Genome, Viral↗
PubMed2003

Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions.

Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively. These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.

Bacteriophage P22↗
PubMed2003

Structure of isolated nucleocapsids from venezuelan equine encephalitis virus and implications for assembly and disassembly of enveloped virus.

Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is approximately 11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.

Encephalitis Virus, Venezuelan Equine↗
PubMed2002

The skeletal muscle Ca2+ release channel has an oxidoreductase-like domain.

We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle Ca2+ release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22A structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [3H]NAD+, supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor.

Amino Acid Sequence↗
PubMed2002

Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum.

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

Adenosine Triphosphatases↗
PubMed2002

High resolution structural studies of complex icosahedral viruses: a brief overview.

Structural descriptions of viral particles are key to our understanding of their assembly mechanisms and properties. We will describe the application of X-ray crystallography and electron cryomicroscopy to the structural determination of the bluetongue virus core and the herpesvirus capsid. These represent the highest resolution structural studies carried out by these techniques on such complex and large icosahedral virus particles. The bluetongue virus core consists of two layers of distinct proteins with different protein packing symmetries, while the herpes virus capsid is made up of four types of proteins with 3.3 MDa per asymmetric unit. The structural results reveal that each of these proteins has distinct folds and they are packed uniquely to form stable particles.

Animals↗
PubMed2002

Bilamellar cationic liposomes protect adenovectors from preexisting humoral immune responses.

Adenoviral vectors have been widely used for gene therapy, but they are limited both by the presence of a humoral immune response that dramatically decreases the level of transduction after reinjection and by their requirement for target cells to express appropriate receptors such as Coxsackie adenovirus receptor (CAR). To overcome both limits, we encapsulated adenovectors using bilamellar DOTAP:chol liposomes. Electron micrography (EM) showed that these liposomes efficiently encapsulated the vectors, allowing CAR-independent adenovector transduction of otherwise resistant cells. DOTAP:chol-encapsulated adenovectors encoding LacZ or alpha(1)-antitrypsin inhibitor (AAT) were also functionally resistant ex vivo and in vivo to the neutralizing effects of human anti-adenoviral antibodies, unlike other liposomal systems. Hence, bilamellar DOTAP:chol liposomes may be useful for applications using adenovectors in which the target cells lack adenoviral receptors or in which the recipient already has or develops a neutralizing antibody response that would otherwise inactivate readministered vector.

Adenoviridae↗
PubMed2002

Deriving folds of macromolecular complexes through electron cryomicroscopy and bioinformatics approaches.

Intermediate-resolution (7-9A) structures of large macromolecular complexes can be obtained by electron cryomicroscopy. This structural information, combined with bioinformatics data for the individual protein components or domains, can lead to a fold model for the entire complex. Such approaches have been demonstrated with the 6.8 A structure of the rice dwarf virus to derive models for the major capsid shell proteins.

Amino Acid Sequence↗
PubMed2001

Quaternary structure of human fatty acid synthase by electron cryomicroscopy.

We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites.

Binding Sites↗
PubMed2001

Web-based Simulation for Contrast Transfer Function and Envelope Functions.

We have implemented a Web-based simulation program for the contrast transfer function and envelope functions in Java and JavaScript. The simulation provides interactive controls of all the parameters in those functions. In addition to the predefined functions, users can easily define new functions that use any of the parameters in the preset functions. The most useful feature of this new simulation program is the convenient, universal accessibility through Web browsers on any computer platform that supports Java, such as Netscape and Internet Explorer.

Journal Article↗