Final Report Summary - PLATELET RECEPTORS (Characterizing Regulatory Principles in Platelet Surface Receptors Via X-ray Crystallography and Small-molecule Design) Blood coagulation in mammals is mediated by platelets, which are activated following damage to the endothelium, as well as in pathological conditions such as interactions with certain toxins or with cancer cells. A key platelet-activating receptor, essential in hemostasis and thrombosis as well as in vascular integrity, is the C-type lectin-like 2 (CLEC-2). In pathophysiology, CLEC-2 mediates snake envenomation via interactions with the snake toxin rhodocytin, especially in Southeast Asia, resulting in massive platelet aggregation and in tissue damage. A more widespread pathophysiological role of CLEC-2 is in promoting cancer metastasis when interacting with the tumor overexpressed transmembrane protein podoplanin, involved in numerous types of cancers. Those interactions induce platelet activation, which in turn provide cancerous cells with growth factor and protection from the immune system, enabling them to form nests in the blood stream. Elucidation and modulation of podoplanin–CLEC-2 interactions is of major importance in our battle against cancer growth and metastasis. Nevertheless, the mode of those interactions is yet ambiguous and no compound has been approved for modulating binding. CLEC-2 exists as monomers and as inactive homodimers on resting platelets, while ligand binding stimulates its oligomerization. It is not clear whether the inactive and active states of CLEC-2 dimer maintain similar protein-protein interfaces. The lack of information about the dimeric interface withholds clear description of CLEC-2 activation mechanism. Accordingly, we sought to shed light on CLEC-2 dimerization interface as well as on its binding site for podoplanin, and modulate these interactions. By utilizing computational methods we first predicted the binding pocket of CLEC-2 to a sialo-sugar moiety decorating podoplanin and known to be essential for their interactions. This was followed by the establishment of facilities for large-scale protein production and purification to confirm our prediction. We managed the challenging expression and purification of CLEC-2 from E.coli inclusion bodies, and determined its X-ray structure in its native form, de-novo to 1.56Å resolution, and in the presence of compounds we designed to imitate the sugar moiety on podoplanin. In the latter X-ray structures, CLEC-2 appeared as a dimer, induced by the presence of these compounds, with our putative binding pocket buried within the dimerization interface. Our newly discovered binding motif of CLEC-2 provides a target for the inhibition of CLEC-2-podoplanin interactions to attenuate cancer growth and metastasis in a specific manner. Both podoplanin and CLEC-2 undergo specific post-translational modifications that are important for their activity and interaction. These modifications can only be obtained by using a mammalian expression system, thus leading us to attempt large-scale protein production in human cell lines. We thus established a fully equipped tissue culture facility designed for large-scale protein production, and optimized DNA constructs, transfection, protein expression and purification conditions with our chosen cell line. Unfortunately, all was attenuated when the growth media of our cell line was taken off the market and all substitutions failed to provide satisfactory cell viability or transfectivity. Back to starting point, we had to screen for new cell lines and conditions, and after encountering many challenges in the process we are now establishing a new large-scale protein production system. This is expected to provide us with high protein yields to reach our goals. Up till now, the low yield and high cost of the mammalian expression system hindered extensive experiments. Nevertheless, we were able to obtain sufficient amounts of CLEC-2 and podoplanin for quality, oligomerization and glycosylation evaluation, and for initial binding affinity measurements and crystallization experiments. We currently work to further improve the production process, in addition to testing enzyme-induced deglycosyaltion methods for increased protein homogeneity while retaining native fold and activity. The project continues beyond the expected period and we aim to complete our binding affinity studies using the native CLEC-2, and subsequent site-directed mutagenesis in our CLEC-2-podoplanin predicted binding pocket. We already completed an in-silico screening of a library of small molecules against this binding pocket, using strict screening criteria, to identify candidates for future drug discovery attempts. These molecules will next be tested for inhibiting CLEC-2 - podoplanin interactions. Characterization and modulation of CLEC-2 is significant on many levels. CLEC-2 represents an example for a protein that in its native form plays roles both in vital physiological processes, as well as in devastating pathologies such as cancer and mediation of snake envenomation. Special care should be taken when modulating such protein, in order to prevent interfering with physiological functions and inducing severe side-effects of potential drugs. This calls for an unambiguous identification of the different binding interfaces of CLEC-2 to its ligands. Therefore, we strive for the atomic level characterization and specific modulation of CLEC-2 interactions with podoplanin, to attenuate cancer growth and metastasis from numerus types of cancers.Reintegration of the Fellow was overall very successful, with the establishment of an independent lab at the Technion – Israel Institute of Technology. With the help of the CIG grant, lab members travel to national and international conferences and attend workshops to enhance their expertise. Making science accessible to the public is also a major goal of the lab, including seminars and press releases (see information in the lab website - http://mlandau.net.technion.ac.il/). The CIG funding was instrumental in developing new methodologies that were extensively applied in the lab, and resulted, for example, in a recent high impact publication (Tayeb-Fligelman et al. Science 355(6327): 831-833; 2017). The support from the CIG was therefore acknowledged in this paper.
