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Crystal structure of native Anopheles gambiae serpin-2, a negative regulator of melanization in mosquitoes

Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
Proteins Structure Function and Bioinformatics (Impact Factor: 3.34). 06/2011; 79(6):1999-2003. DOI: 10.1002/prot.23002
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    ABSTRACT: Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits CLIPB9, which functions in a serine proteinase cascade culminating in the activation of pro-phenoloxidase (proPO) and melanization. Silencing of SRPN2 in An. gambiae results in spontaneous melanization and decreased lifespan and is therefore a promising target for vector control. The previously determined structure of SRPN2 revealed a partial insertion of the hinge region of the reactive center loop (RCL) into β sheet A. This partial hinge insertion participates in heparin-linked activation in other serpins, notably antithrombin III. SRPN2 does not contain a heparin binding site and any possible mechanistic function of the hinge insertion was previously unknown. To investigate the function of the SRPN2 hinge insertion, we developed three SRPN2 variants in which the hinge regions are either constitutively expelled or inserted and analyzed their structure, thermostability, and inhibitory activity. We determined that constitutive hinge expulsion resulted in a 2.7-fold increase in the rate of CLIPB9Xa inhibition, which is significantly lower than previous observations of allosteric serpin activation. Furthermore, we determined that stable insertion of the hinge region did not appreciably decrease the accessibility of the RCL to CLIPB9. Together, these results indicate the partial hinge insertion in SRPN2 does not participate in the allosteric activation observed in other serpins and instead represents a molecular trade-off between RCL accessibility and efficient formation of an inhibitory complex with the cognate proteinase.
    Journal of Biological Chemistry 12/2014; 290(5). DOI:10.1074/jbc.M114.625665 · 4.60 Impact Factor
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    ABSTRACT: Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities; particularly those in which protease mediated cascades are active. The Drosophila serpin, Spn43Ac, negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus, Beauveria bassiana, offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and recalcitrant (due to strong immune responses) targets. Since Spn43Ac acts to inhibit Toll mediated activation of defense responses, we explored the feasibility of a new strategy to engineer increased virulence of entomopathogenic fungi by expression of Spn43Ac in the fungus. Compared to the wild type parent, the LD50 of B. bassiana expressing Spn43Ac (Bb::S43Ac-1) was reduced ∼3-fold and the LT50 decreased by ∼24% against the Greater Wax moth, Galleria mellonella, with more rapid proliferation of hyphal bodies seen in the host haemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin > laminarin > lipopolysachharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassiana derived cuticle degrading protease, CDEP-1. These results expand our understanding of Spn43Ac function and confirmed that suppression of insect immune system defenses represents a feasible approach at engineering entomopathogenic fungi for greater efficacy.
    Applied and Environmental Microbiology 05/2014; 80(15). DOI:10.1128/AEM.01197-14 · 3.95 Impact Factor
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    ABSTRACT: 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 CLIPB9Xa 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.
    Entomological Society of America Annual Meeting 2014; 11/2014

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