Biomedical subjects

David K Cole

Publications and source records attributed to David K Cole.

11 recordsLinked to original sources

PubMed2006

Crystal structure of HLA-A*2402 complexed with a telomerase peptide.

HLA-A*2402 is the most commonly expressed HLA allele in oriental populations. It is also widely expressed in the Caucasian population, making it one of, if not the most abundant HLA I types. In order to study its structure in terms of overall fold and peptide presentation, a soluble form of this HLA I (alpha1, alpha2, alpha3 and beta(2)m domains) has been expressed, refolded and crystallized in complex with a cancer-related telomerase peptide (VYGFVRACL), and its structure has been solved to 2.8 A resolution. The overall structure of HLA-A*2402 is virtually identical to other reported peptide-HLA I structures. However, there are distinct features observable from this structure at the HLA I peptide binding pockets. The size and depth of pocket B makes it highly suitable for binding to large aromatic side chains, which explains the high prevalence of tyrosine at peptide position 2. Also, for HLA binding at peptide position 5, there is an additional anchor point, which allows the proximal amino acids to protrude out, providing a prominent feature for TCR interaction. Finally, pocket F allows the anchor residue at position 9 to be bound unusually deeply within the HLA structure.

Amino Acid Sequence↗

PubMed2005

Crystallization and preliminary X-ray structural studies of a high-affinity CD8alphaalpha co-receptor to pMHC.

The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR-pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6(4)22 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 A. VM calculations indicated one molecule per asymmetric unit. A 2 A data set was collected and the structure is currently being determined using molecular replacement.

CD8-Positive T-Lymphocytes↗

PubMed2004

Characterization of the heptad repeat regions, HR1 and HR2, and design of a fusion core structure model of the spike protein from severe acute respiratory syndrome (SARS) coronavirus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emergent virus responsible for a worldwide epidemic in 2003. The coronavirus spike proteins belong to class I fusion proteins, and are characterized by the existence of two heptad repeat (HR) regions, HR1 and HR2. The HR1 region in coronaviruses is predicted to be considerably longer than that in other type I virus fusion proteins. Therefore the exact binding sequence to HR2 from the HR1 is not clear. In this study, we defined the region of HR1 that binds to HR2 by a series of biochemical and biophysical measures. Subsequently the defined HR1 (902-952) and HR2 (1145-1184) chains, which are different from previously defined binding regions, were linked together by a flexible linker to form a single-chain construct, 2-Helix. This protein was expressed in Escherichia coli and forms a typical six-helix coiled coil bundle. Highly conserved HR regions between mouse hepatitis virus (MHV) and SARS-CoV spike proteins suggest a similar three-dimensional structure for the two fusion cores. Here, we constructed a homology model for SARS coronavirus fusion core based on our biochemical analysis and determined the MHV fusion core structure. We also propose an important target site for fusion inhibitor design and several strategies, which have been successfully used in fusion inhibitor design for human immunodeficiency virus (HIV), for the treatment of SARS infection.

Amino Acid Sequence↗

PubMed2004

Biochemical, biophysical and preliminary X-ray crystallographic analyses of the fusion core of Sendai virus F protein.

It is emerging that enveloped viruses may adopt a unique entry/fusion mechanism; in paramyxoviruses, including Sendai virus (SeV), the attachment protein HN (or its homologue H or G) binds a cellular receptor which triggers conformational changes of its fusion protein, F. There are at least three conformations of the F protein in the current fusion model: the pre-fusion native conformation, the pre-hairpin intermediate conformation and the post-fusion coiled-coil conformation. The fusion mechanism of SeV, a member of the Paramyxoviridae family, has been well established and several structural and functional domains or modules have been proposed from studies of its F protein. However, biochemical and biophysical studies of the heptad-repeat (HR) regions (HR1 and HR2) have not been systematically carried out. HR1 and HR2 strongly interact with each other to form a stable six-helix coiled-coil bundle as the post-fusion conformation. In this study, a single-chain HR1-linker-HR2 protein of SeV was prepared in an Escherichia coli expression system and biochemical and biophysical analyses showed it to form a typical six-helix coiled-coil bundle; its trigonal crystals diffracted X-rays to 2.5 A resolution. The crystal structure will help to reveal the structural requirements of the post-fusion coiled-coil conformation of SeV F protein.

Amino Acid Sequence↗

PubMed2004

Complex assembly, crystallization and preliminary X-ray crystallographic studies of MHC H-2Kd complexed with an HBV-core nonapeptide.

In order to establish a system for structural studies of the murine class I major histocompatibility antigen complex (MHC) H-2Kd, a bacterial expression system and in vitro refolding preparation of the complex of H-2Kd with human beta2m and the immunodominant peptide SYVNTNMGL from hepatitis B virus (HBV) core-protein residues 87-95 was employed. The complex (45 kDa) was crystallized; the crystals belong to space group P222(1), with unit-cell parameters a = 89.082, b = 110.398, c = 47.015 A, alpha = beta = gamma = 90 degrees. The crystals contain one complex per asymmetric unit and diffract X-rays to at least 2.06 A resolution. The structure has been solved by molecular replacement and is the first crystal structure of a peptide-H-2Kd complex.

Amino Acid Sequence↗

PubMed2004

Following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a newly identified member of Family Coronaviridae. Coronavirus envelope spike protein S is a class I viral fusion protein which is characterized by the existence of two heptad repeat regions (HR1 and HR2) (forming a complex called fusion core). Here we report that by using in vitro bio-engineering techniques, SARS-CoV HR1 and HR2 bind to each other and form a typical 6-helix bundle. The HR2, either as a synthetic peptide or as a GST-fusion polypeptide, is a potent inhibitor of virus entry. The results do show that SARS-CoV follows the general fusion mechanism of class I viruses and this lays the ground for identification of virus fusion/entry inhibitors for this devastating emerging virus.

Circular Dichroism↗

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

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

SARS coronavirus induces apoptosis in Vero E6 cells.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease. Its etiological agent has been convincingly identified as a new member of family Coronaviridae (SARS-CoV). It causes serious damage to the respiratory system yet the mechanism is not clear. Infection-induced apoptosis or necrosis is suspected but no direct evidence for this yet exists. To date, Vero E6 cells are the only cell line that could be used to replicate the virus with obvious CPE (cytopathic effect) in vitro. It is known for some viruses (including members of family Coronaviridae) that CPE can be caused either by virus-induced apoptosis (active death) or cell necrosis (passive death). In this study, we examined the apoptosis in the SARS-CoV infected Vero E6 cells. Indeed, the results do show that the CPE was induced by apoptosis rather than necrosis, shown by typical DNA fragmentation, through the existence of apoptotic bodies and swollen mitochondria. This observation has some implications for the SARS-CoV pathogenicity: SARS-CoV does induce apoptosis in cell cultures and might have the same effect in vivo, responsible for the severe damage of the respiratory system.

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

CD8: adhesion molecule, co-receptor and immuno-modulator.

CD8 is a cell surface glycoprotein found in cytotoxic T lymphocytes, which are important components in cellular immunity, esp. in the immune response to cancer and chronic infections. There are two forms of CD8, either as an alphaalpha homodimer or alphabeta heterodimer. It acts as an "assistant" or co-receptor in the function of cytotoxic T cells where specific immunity is mediated by interaction of specific T cell receptor (alphabeta TCR) and its ligand peptide major histocompatibility complex (pMHC). CD8 also binds to pMHC but away from the interface of pMHC and TCR contact, thereof no influence on the specificity of this interaction. If the TCR and CD8 bind to the same pMHC at the same time, CD8 is defined as a co-receptor, functioning through its signalling via its cytoplasmic tyrosine phosphorylation pathway; if CD8 binds to pMHC independently of the TCR, it is defined as an adhesion molecule. At present, the co-receptor function theory is dominated in the field. Recent study has also shown that murine CD8 alphaalpha binds to TL antigen, an MHC homologue, therefore acts as an immuno-modulator. In this review, we discuss these current understandings of the three aspects of the CD8 functions and their structural basis.

Animals↗