The thrombin mutant D221A/D222K (ARK) does not bind Na+ and has interesting
functional properties intermediate between those of the slow and fast forms of
wild type. We solved the X-ray crystal structure of ARK bound at exosite I with
a fragment of hirudin at 2.4-A resolution. The structure shows a slight collapse
of the 186 and 220 loops into the Na+ binding site due to disruption of the
Asp222:Arg187 ion-pair. The backbone O atoms of Arg221a and Lys224 are shifted
into conformations that eliminate optimal interaction with Na+. A paucity of
solvent molecules in the Na+ binding site is also noted, by analogy to what is
seen in the structure of the slow form. These findings reinforce the crucial
role of the Asp222:Arg187 ion-pair in stabilizing the fast form of thrombin.
