title: Inhibitory activity of the Drosophila melanogaster serpin Necrotic is dependent on lysine residues in the D-helix.
abstract: Necrotic is a member of the serine protease inhibitor or serpin superfamily. It is a potent inhibitor of elastase and chymotrypsin type proteases and is responsible for regulating the anti-fungal response in Drosophila melanogaster. Necrotic contains three basic lysine residues within the D-helix that are homologous to those found in the heparin-binding domain of antithrombin and heparin co-factor II. We show here that substitution of all three lysine residues for glutamines caused cellular necrosis and premature death in Drosophila in keeping with a loss of function phenotype. The lysine to glutamine substitutions had no effect on the overall structure of recombinant Necrotic protein but abolished the formation of stable complexes with target proteases. Individual substitutions with either glutamine or alanine demonstrated that lysine 68 was the most critical residue for inhibitory activity. Despite the homology to other serpins, Necrotic did not bind, nor was it activated by sulfated glycans. These data demonstrate a critical role for basic residues within the D-helix (and lysine 68 in particular) in the inhibitory mechanism of the serpin Necrotic.
authors.affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Robertson
authors.forename: Didier
authors.initials: D
authors.lastname: Belorgey
authors.forename: David
authors.initials: D
authors.lastname: Gubb
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Dafforn
authors.forename: David A
authors.initials: DA
authors.lastname: Lomas
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C417282:nec protein, Drosophila; D006493:Heparin; D008239:Lysine