Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin

Abstract

Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the 4th, 5th, and most of the 6th EGF-like domain (TM456m) has been prepared that has much improved solubility, thrombin-binding capacity, and anticoagulant activity over previous TM456 constructs. In the present work we compare backbone amide exchange of human α-thrombin in three states: apo, PPACK-bound, and TM456m-bound. Beyond causing decreased amide exchange at their binding sites, TM and PPACK both cause decreased amide exchange in regions more distant from the active site, within the γ-loop and the adjacent N-terminus of the heavy chain. The decreased amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, HDXMS results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.

NMR reveals a dynamic allosteric pathway in thrombin

“…Previously published protease sensitivity and amide exchange studies consistently showed that the γ-loop is much more dynamic in apo-thrombin than in PPACK thrombin1536. Therefore it was not surprising that most of the NH cross peaks for γ-loop residues were missing, cross peaks for residues V138(174) in the β-strand leading into the γ-loop, and V158(199) and N159(200) in the β-strand exiting the γ-loop showed μs-ms motions in apo-thrombin (Supplementary Fig.…”

“…3). Recent HDXMS data showed a dramatic decrease in amide exchange in the N-terminus of the heavy chain upon PPACK-binding, however, some amides still exchanged within residues 16–33(37–44), indicating that part of this region is still exposed and/or dynamic15. The NMR results presented here now allow us to localize the change in dynamics to the most N-terminal residues 16–18 (37–39) of the thrombin heavy chain and to conclude that residues subsequent to these remain dynamic.…”

“…However, the core structure of thrombin with different effectors and/or with or without bound substrate, do not reveal significant conformational changes. On the other hand, hydrogen-deuterium exchange followed by mass spectrometry showed rapid exchange of many of the backbone amides in thrombin, particularly in the surface loops1415 and NMR experiments showed missing resonances1112 both suggesting that thrombin may be dynamic.…”

Dynamic Consequences of Mutation of Tryptophan 215 in Thrombin

“…The TM activation of thrombin towards protein C cleavage was first performed by incubating the TM construct TM456m with purified human α-thrombin before adding protein C (Hematologic Technologies, Essex Junction, VT). TM456m was purified as previously described (14). Following a 20 min incubation with protein C, the thrombin was inactivated by addition of heparin-antithrombin-III, and the activated protein C was assayed by the addition of S-2366.…”

Ligand binding modulates the structural dynamics and activity of urokinase-type plasminogen activator: A possible mechanism of plasminogen activation

“…Five peptides corresponded to the 140s loop (residues 136–148, MH + 1401.58Da; residues 138–148, MH + 1169.53Da; residues 149–157, MH + 1169.63Da; residues 149–159, MH + 1355.73Da; residues 150–159, MH + 1192.67Da). The 140s loop is known as a very flexible loop in trypsin-like serine proteases, and it has been shown that binding of active site ligands allosterically reduces the conformational flexibility of this loop [ 24 ]. In good agreement with this notion, our results revealed that all five peptides covering the 140s loop showed decreased deuterium incorporation upon binding of EGR-cmk (Figs 3C and S1 ).…”