Biomedical subjects

Christopher A O'Callaghan

Publications and source records attributed to Christopher A O'Callaghan.

4 recordsLinked to original sources

PubMed2006

The crystal structure and mutational binding analysis of the extracellular domain of the platelet-activating receptor CLEC-2.

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.

Crystallization↗

PubMed2005

Crystallization and X-ray diffraction analysis of human CLEC-2.

The human C-type lectin-like protein CLEC-2 has recently been shown to be expressed on the surface of platelets and to function as a receptor for the snake-venom protein rhodocytin. The C-type lectin-like domain (CTLD) of CLEC-2 was expressed in Escherichia coli, refolded and purified. Crystals of this recombinant CLEC-2 were grown by sitting-drop vapour diffusion using polyethylene glycol (PEG) 6000 as a precipitant. After optimization, crystals were grown which diffracted to 2.0 A using in-house radiation (lambda = 1.5418 A). These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.407, b = 55.143, c = 56.078 A. The presence of one molecule per asymmetric unit is consistent with a crystal volume per unit weight (VM) of 1.82 A3 Da(-1) and a solvent content of 32.6%. These results suggest that crystals producing diffraction of this quality will be suitable for the structural determination of human CLEC-2.

Blood Platelets↗

PubMed2002

Tetrameric complexes of HLA-E, HLA-F, and HLA-G.

HLA-E, HLA-F, and HLA-G are human nonclassical MHC class Ib molecules. To study the function and identify potential ligands of these molecules, we constructed tetrameric complexes. In this brief review, we discuss the methods used to produce such tetramers and the interesting results they provided. HLA-E tetramers bound to natural killer (NK) cells and T cells, allowing the identification of CD94/NKG2 molecules as receptors for HLA-E. HLA-G tetramers interacted with immunoglobulin-like transcript-2 (ILT2) and ILT4 receptors, aiding the understanding of HLA-G function during pregnancy. Tetrameric complexes of HLA-F also bound to ILT2 and ILT4.

Amino Acid Sequence↗

PubMed2002

Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D.

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.

Animals↗