Crystal structure of the thrombin mutant D221A/D222K: the Asp222:Arg187 ion-pair stabilizes the fast form |
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Authors: | Pineda Agustin O Zhang Erli Guinto Enriqueta R Savvides Savvas N Tulinsky Alexander Di Cera Enrico |
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Institution: | Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110, USA. |
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Abstract: | 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. |
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