title: Structural and inhibitory effects of hinge loop mutagenesis in serpin-2 from the malaria vector Anopheles gambiae.
abstract: Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9), which functions in a serine proteinase cascade culminating in the activation of prophenoloxidase and melanization. Silencing of SRPN2 in A. gambiae results in spontaneous melanization and decreased life span 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 that 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.
authors.affiliation: From the Division of Biology, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: From the Division of Biology, Kansas State University, Manhattan, Kansas 66506.
authors.forename: David A
authors.initials: DA
authors.lastname: Meekins
authors.affiliation: From the Division of Biology, Kansas State University, Manhattan, Kansas 66506, the Department of Entomology, College of Agriculture and Biotechnology, China Agricultural University, Beijing, China.
authors.forename: Chunju
authors.initials: C
authors.lastname: An
authors.affiliation: the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Michal
authors.initials: M
authors.lastname: Zolkiewski
authors.affiliation: Industrial Macromolecular Crystallography Association Collaborative Access Team, Hauptman-Woodward Medical Research Institute, Advanced Photon Source Argonne National Laboratory, Argonne, Illinois 60439, and.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Battaile
authors.affiliation: the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kanost
authors.affiliation: the Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas, Lawrence, Kansas 66407.
authors.forename: Scott
authors.initials: S
authors.lastname: Lovell
authors.affiliation: From the Division of Biology, Kansas State University, Manhattan, Kansas 66506, kmichel@ksu.edu.
authors.forename: Kristin
authors.initials: K
authors.lastname: Michel
mesh_terms: D000818:Animals; D000852:Anopheles / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D018360:Crystallography, X-Ray; D016296:Mutagenesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D015843:Serpins; C434683:serpin-2
keywords: Crystal Structure; Host-Pathogen Interaction; Insect Immunity; Malaria; Serpin; Structural Biology