Agustin O. Pineda

The Thrombin Epitope Recognizing Thrombomodulin Is a Highly Cooperative Hot Spot in Exosite I

The functional epitope of thrombin recognizing thrombomodulin was mapped using Ala-scanning mutagenesis of 54 residues located around the active site, the Na+ binding loop, the 186-loop, the autolysis loop, exosite I, and exosite II. The epitope for thrombomodulin binding is shaped as a hot spot in exosite I, centered around the buried ion quartet formed by Arg67, Lys70, Glu77, and Glu80, and capped by the hydrophobic residues Tyr76 and Ile82. The hot spot is a much smaller subset of the structural epitope for thrombomodulin binding recently documented by x-ray crystallography. Interestingly, the contribution of each residue of the epitope to the binding free energy shows no correlation with the change in its accessible surface area upon formation of the thrombin-thrombomodulin complex. Furthermore, residues of the epitope are strongly coupled in the recognition of thrombomodulin, as seen for the interaction of human growth hormone and insulin with their receptors. Finally, the Ala substitution of two negatively charged residues in exosite II, Asp100 and Asp178, is found unexpectedly to significantly increase thrombomodulin binding.

Crystal structure of thrombin in a self-inhibited conformation.

The activating effect of Na+ on thrombin is allosteric and depends on the conformational transition from a low activity Na+-free (slow) form to a high activity Na+-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na+ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na+ site and the active site regions. Here, we report a 1.87Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow → fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na+-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na+ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

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

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 αvβ3 and α5β1 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.

Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery.

Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191–Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an ∼100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-Å resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH2Cl, as revealed by the 1.8-Å resolution structure of the complex. We conclude that the Cys-191–Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation.