Abstract
Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.
Highlights
Binding of Naϩ to human thrombin results in enhanced catalytic activity due to faster substrate diffusion into the active site and faster acylation [1,2,3]
The lack of Naϩ activation in human thrombin would produce an enzyme with little catalytic activity toward fibrinogen and PAR1, incompatible with biological function [14]
In addition to the basic differences uncovered in the role of PARs in platelet activation between mice and humans [44, 47], thrombin in the mouse lacks Naϩ activation [23], which is a hallmark feature of the human enzyme [2]
Summary
Binding of Naϩ to human thrombin results in enhanced catalytic activity due to faster substrate diffusion into the active site and faster acylation [1,2,3]. Murine thrombin has solved this conundrum by mimicking functionally the Naϩ-bound fast form of the human enzyme, thereby retaining high activity toward fibrinogen, PAR1, and PAR4 and without compromising activation of protein C [23]. Such molecular mimicry of Naϩ activation may have turned beneficial during evolution to counter the effect of numerous mutations that in humans destabilize Naϩ binding and cause bleeding (24 –29). The thrombin chimera was constructed by replacing residues 184a, 186, 186a, 186b, 186c, and 222 in human thrombin with those of the murine enzyme
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