The Crystal Structure and Mutational Binding Analysis of the Extracellular Domain of the Platelet-activating Receptor CLEC-2

Aleksandra A. Watson,Christopher A. O'Callaghan,Johannes A. Eble,Thomas S. Walter,James Brown,Karl Harlos

doi:10.1074/jbc.m610383200

Abstract

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.

Highlights

Platelet activation at sites of vascular injury is critical for primary hemostasis, but can trigger arterial thrombosis in vascular disease
The basic architecture of CLEC-2 preserves key features of the C-type lectin-like domain, with two antiparallel ␤-sheets flanked by two ␣-helices (Fig. 2A and supplementary Fig. S1)
The structure was compared with related receptors, which bind protein ligands and for which detailed ligand binding data is available: the oxidized low density lipoprotein (LDL) receptor LOX-1 and the immune receptors NKG2D and Ly49C, which bind major histocompatibility complex (MHC) class I-like molecules (18 –20)

Summary

EXPERIMENTAL PROCEDURES

CLEC-2 Crystallization, Data Collection, and Structure Determination—The extracellular domain of CLEC-2 from residues 96 to 221 was cloned, expressed, purified, and crystallized as described previously (7, 8). The ␣␤-rhodocytin dimer was manually assembled by superimposing the ␣and ␤-subunits of rhodocytin onto a template of the related C-type lectin snake venom protein Aa-X-Bp-I using Coot This model of the ␣␤-rhodocytin dimer was regularized using energy minimization algorithms implemented by CNS solve (12). Ranked complexes were subjected to van der Waals minimization using CHARMM to remove potential side chain clashes (see Supplementary Materials for more details of the docking method) (30) To these ClusPro-validated complexes, a set of biological selection criteria was applied to select the most plausible model. Interacting faces were matched on the basis of shape complementarity, surface hydrophobicity, electrostatic compatibility and the location of the CLEC-2 membrane insertion point From this model of the interaction, and from our previous knowledge of the binding properties of C-type lectin-like molecules, CLEC-2 residues were selected for mutational studies to determine their involvement in ligand binding (31).

Crystallographic data collection

Ramachandran values

RESULTS

DISCUSSION