Thrombin Functions through Its RGD Sequence in a Non-canonical Conformation

Abstract

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with ␣ v ␤ 3 and ␣ 5 ␤ 1 integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Å resolution crystal structure of free thrombin grown in the presence of high salt (400 mM KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Å away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.

Crystal Structure of Thrombin in a Self-inhibited Conformation

“…Minor van der Waals' interactions involve the 60-loop from Tyr-60a to Asp-60e but do not extend to Trp-215. The structure does not feature the active site occlusion by Glu-192 reported in recent structures of inactive thrombin (13,16,17), nor does it exhibit perturbation of the 60 loop (12,17) or significant exposure of the RGD sequence (15). We have shown that the shift in the position of Glu-192 is an artifact of crystal packing.…”

“…The same conclusion applies to perturbation of the 60 loop, for which mutagenesis studies have ruled out any linkage with Na ϩ binding (9). However, exposure of the RGD sequence is well established functionally (15) and is confirmed by the structure of the mutant D189A, 2 where packing 2 C. J. Carrell, F. S. Mathews, and E. Di Cera, manuscript in preparation. …”

“…The inactive conformation reported in this structure is largely the result of the drastic charge reversal introduced near the Na ϩ site and significant crystal packing. The recent structure of the thrombin mutant R77aA in the presence of KCl contains two molecules in the asymmetric unit, one with K ϩ bound to the Na ϩ site (molecule 1) and the other (molecule 2) in an inactive conformation with the Na ϩ site free (15,16). This structure too contains substantial packing interactions in the asymmetric unit that affect a surface area Ͼ600 Å ( Table 2).…”

“…Some of these structures (13,16,17) have been claimed to capture the essential properties of the inactive slow form S*, with the active site occluded and the Na ϩ site disordered. One structure has suggested that the physiologic role of the inactive slow form of thrombin could be to expose the RGD sequence (15) for interaction with integrins (18). Current structural evidence of the existence of an inactive slow form is interesting and potentially relevant to thrombin allostery.…”

“…The inactive S* form was found incapable of binding Na ϩ and substrate at the active site (10). Following the pioneering structure of thrombin free of inhibitors and Na ϩ portraying the active slow form (9,11), a number of crystal structures of free thrombin have been obtained with the enzyme in various inactive forms (12)(13)(14)(15)(16)(17). Some of these structures (13,16,17) have been claimed to capture the essential properties of the inactive slow form S*, with the active site occluded and the Na ϩ site disordered.…”