The platelet receptor CLEC-2 is active as a dimer

Watson AA, Christou CM, James JR, Fenton-May AE, Moncayo GE, Mistry AR, Davis SJ, Gilbert RJ, Chakera A, O’Callaghan CA. (2009), Biochemistry. 48, 10988-96

Abstract

The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.

Key figure: Model dimeric CLEC-2 is flexible, and this may enhance ligand binding.

Molecular dynamics analysis of a model of dimeric CLEC-2. (A) Model dimeric CLEC-2 is represented as a cartoon where the individual monomer chains are colored blue and red. Disulfide bonds formed between cysteine residues are marked in yellow, and the putative ligand binding site is represented by a green oval. (B, C) Porcupine plots of the principal mode of conformational variability of the Cα atoms calculated from a CONCOORD ensemble are presented with each blue cone indicating the direction of motion of the atom and the lengths indicating the relative amplitude of the motion. Panel C is related to panel B by a 90° clockwise rotation about the y-axis. Black arrows indicate opposing motions of the two C-type lectin-like domains, demonstrating how the bimodal pattern of motion may allow spreading of the ligand binding region to enhance the ligand interaction.