NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

Abstract

The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na þ have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na þ -binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.allostery | hemostasis | protease | structure | zymogen B lood coagulation (hemostasis) is the result of a cascade of events where zymogens are converted to active proteases through the specific action of a preceding protease (1, 2). This process is tightly regulated to ensure that stable blood clots form rapidly at the site of tissue damage. Dysregulation by several mechanisms is the cause of bleeding disorders, including hemophilia, and of thrombosis, the most common cause of morbidity and mortality in the industrialized world. Most hemostatic proteases have only a single target and are regulated by a single cofactor. However, thrombin (Fig. 1), the final protease in the cascade, has over a dozen substrates and at least five cofactors (3). Thrombin is critical for the initiation, propagation, and attenuation of the hemostatic response, and in each of these phases the activity of thrombin is directed by cofactors (4, 5). Of principal importance are the cofactors that convert thrombin between pro and anticoagulant activities. Thrombomodulin (TM) is an integral membrane protein that alters thrombin specificity from the procoagulant substrates such as PAR1, factors V and VIII, and fibrinogen to specific activation of the anticoagulant protease protein C (6). This change of function is thought to be due to the bloc...

Structural basis for complement factor I control and its disease-associated sequence polymorphisms

“…Such a mechanism is consistent with the observation that intact fI will not cleave C3b in the absence of cofactor, whereas purified SP domain does, albeit with altered specificity and low activity (25). The mechanism is also analogous to the regulation of serine proteases such as factor VIIa (34,35) and thrombin (23), which have allosteric regulator binding sites at locations distant from the zymogenactivation domain or active site (SI Appendix, Fig. S6).…”

“…2C). The heavy-chain N terminus and LDLRA2 domain also form one of the two interfaces between the heavy and light chains, the second being located around the interchain disulfide bridge Cys309-Cys435 (23). Each chain buries about 720 Å 2 of surface in this interface (24).…”

“…Limited proteolysis of native fI under physiological conditions results in cleavage of the SP domain at residues flexible in our structure (26), including the five N-terminal residues that would be buried in the protease in its fully active form, suggesting that the crystal structure is representative of the structure in solution. However, it is very unusual to observe a zymogen-like structure for a processed serine protease in a crystal, and it has been proposed that packing into the crystal environment induces order in loops that are unstructured in solution studies (23).…”

“…Although fI is extreme in the zymogen-like nature of the proteolytically processed form of the enzyme, it is related most closely in terms of activity and regulation to allosterically regulated serine proteases such as thrombin, which recently has been demonstrated to have a dynamic zymogen-activation domain in solution that reorders in the presence of allosteric activators or substrate (23,27). High concentrations of peptide substrates, in the absence of cofactor, can induce low levels of fI activity (28,29), and substrate analog inhibitors also can bind in the absence of either C3b or cofactor (28,30).…”

NMR reveals novel mechanisms of protein activity regulation

“…[33][34][35] Numerous examples are currently available highlighting the inextricable link between protein dynamics and allostery 8,36 . Long-range allosteric coupling between sites through changes in internal dynamics were seen between the nucleotide-binding cleft and the preprotein-binding site in SecA ATPase, 37 between the coordination Na þ site and exosite I in thrombin that regulates enzyme's specificity, 38 between the substrate-binding site and distal loop in dihydrofolate reductase, 39 between the mixed lineage leukemia)-and c-Myb binding sites of the KIX domain of CREB-binding protein, 40 in PDZ signaling domains, 41 the serine protease inhibitor eglin c, 42 upon binding of barstar to the RNase barnase, 43 in the interaction between the Rho GTPasebinding domain and Rac1, 44 upon cyclic nucleotide binding to the exchange protein activated by cAMP, 45,46 a in V-type allosteric enzyme, 47 and in protein kinase A, 48,49 only to mention few recent examples from a long list of systems characterized over the years. Dynamic changes in these systems were accompanied by varying extent of structural changes, ranging from minimal to substantial.…”