Crystal structure of the thrombin mutant D221A/D222K: the Asp222:Arg187 ion-pair stabilizes the fast form.
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.
Pineda
Agustin O
AO
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110, USA.
Zhang
Erli
E
Guinto
Enriqueta R
ER
Savvides
Savvas N
SN
Tulinsky
Alexander
A
Di Cera
Enrico
E
D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D015951:Factor Xa; D008958:Models, Molecular; D018716:Molecular Mimicry; D020125:Mutation, Missense; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics
D012964:Sodium; D013917:Thrombin; D015951:Factor Xa