|
The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with
sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4
desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined
and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and
0.163, respectively. The binding of hirugen to thrombin is similar to that of
the binding of the C-terminal dodecapeptide of hirudin, including that of the
terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of
sulfation, increases the binding affinity by an order of magnitude, is involved
in an extended hydrogen bonding network utilizing all three sulfato oxygen
atoms. The hirugen-thrombin complex is the first thrombin structure determined
to have an unobstructed active site; this site is practically identical in
positioning of catalytic residues and in its hydrogen bonding pattern with that
of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is
cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1
occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like
that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4
spacer to hirugen is disordered in the structure, as is the 3(10) turn of
hirugen. The latter must be related to the simultaneous absence both of
sulfation and of the last residue of hirudin (Gln65'). In addition, the
autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures.
Changes in circular dichroism upon hirugen binding are therefore most likely the
result of the flexibility associated with this loop.
|
