Kristin Michel photoKristin Michel  (2009-2011)
Associate Professor, Division of Biology
Kansas State University

Structure and Target Protease of Anopheles SRPN6, an Inhibitor of Malaria Parasite Infection (ARRA)

Despite many control attempts, malaria continues to be a major health problem (World health report, WHO 2002). Functional genomic and reverse genetic analyses of mosquito/parasite interactions have identified several parasite antagonists, including a serine protease inhibitor, serpin-6 (SRPN6) from the African mosquito Anopheles gambiae, that negatively affect parasite transmission. Serpins inhibit their target proteases very selectively via an exposed loop near the carboxyl-terminal end of the serpin sequence. Proteolytic cleavage of the serpin results in covalent trapping of the protease, forming complexes that are readily detectable by Western analysis after SDS-page. Identifying the protease target(s) of SRPN6 and elucidating the structure of the complex and/or its protease target(s), could lead to a new strategy for malaria control via small-molecule protease inhibitors. The specific aims outlined below are designed to pursue this hypothesis.

Aim 1: Analysis of the crystal structure of SRPN6 in its native conformation. We already have produced recombinant, functional SRPN6 in soluble form and, working with the COBRE Protein Purification Core Lab, we will optimize its large scale purification for crystallization studies. All steps of crystallization and X-ray analysis will be performed in collaboration with the COBRE Protein Structure Core Lab. Structures determined will be compared to those of other known serpins because structural similarity often transcends a lack of sequence similarity among functionally related proteins.

Aim 2: Identification of target serine protease(s) that are inhibited by SRPN6. Antibodies to serpins will be used to isolate serpin-protease complexes formed in malaria-infected mosquitoes, and the protease components in such complexes will be identified by LC/MS/MS analysis of their tryptic peptides.