Mutagenesis analysis of the partial hinge insertion of the reactive center loop of an insect serpin from Anopheles gambiae

Serpin 2 (SRPN2) is a key negative regulator of the serine proteinase cascade that controls activation of prophenoloxidase (proPO) and thus melanization by inhibiting CLIP domain serine proteinase B9 (CLIPB9) in malaria mosquito Anopheles gambiae. An unusual feature of native SRPN2 is the partial insertion of hinge region of the reactive center loop (RCL) into β sheet A, which is only seen in three other mammalian and one beetle serpin. To determine whether this partial insertion of the hinge region contributes to target proteinase inhibition, two mutated forms of SRPN2 were produced in which the serine residue at residue 358 was mutated to a glutamate (S358E) or a tryptophan (S358W). In one of the variants, S358E, but not S358W, the hinge region was expelled out from β sheet A as revealed by x-ray crystallography. However, in vitro inhibition assays with CLIPB9_Xa show that the stoichiometry of inhibition (SI) and second order rate constant (ka) of the variant S358E are comparable to that of the wild-type SRPN2.  These data suggest that in contrast to mammalian serpins such as anti-thrombin III, complete hinge expulsion may not be required for optimal proteinase inhibition. An alternative explanation is that hinge expulsion does not require co-factors, such as sulfated glycosaminoglycans, and is instead mediated by exosite interaction with CLIPB9 prior to cleavage of the scissile bond. The latter proposes a previously unknown mechanism of target selectivity in serpins, which may regulate melanization in insects.