title: Structure-function relationship of SW-AT-1, a serpin-type protease inhibitor in silkworm.
abstract: Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10-5 M-1s-1, for rSW-AT-1 and chymotrpsin is 2.85×10-6 M-1s-1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4-10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D002918:Chymotrypsin; D014357:Trypsin