Abstract
In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 A. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.
Highlights
Senescence stress induces the expression of the cysteine vacuolar proteases, vacuolar processing enzyme-␥ (VPE␥) and RESPONSIVE TO DESICCATION-21 (RD21) [24], and the processing of RD21 into the mature active form [25]
We show that AtSerpin1 can form SDSstable complexes in a manner that is sensitive to the addition of the potent cysteine protease inhibitor, E-64
Clear electron density was observed for all residues in the serpin sequence, including the reactive center loop (RCL)
Summary
Determination of AtSerpin Structure—The full-length AtSerpin cDNA was obtained via The Arabidopsis Information Resource and was cloned into a pET100/D-TOPO expression vector (Invitrogen) whereby the protein was equipped with an N-terminal His tag. Proteins of 14-day-old seedlings were extracted by grinding the whole plants with a mortar and pestle and mixing with extraction buffer (20 mM Tris, pH 8.0, 1 mM EDTA and 50 mM NaCl), followed by centrifugation (15 min, 17,000 ϫ g). The extract (0.3 mg) was labeled in 0.125-ml total volume, containing 50 mM sodium acetate buffer, pH 6, 10 mM dithiothreitol, and 2 M DCG-04. In Vitro Binding—Plants of 14-day-old seedlings, AtSerpin1ko and RD21-ko, were extracted in binding buffer (50 mM sodium acetate, pH 6.0, 1 mM EDTA) and incubated at room temperature for 30 min with 3 mM dithiothreitol and in the presence or absence of recombinant AtSerpin. Samples were analyzed by nonreducing SDS-PAGE and immunoblotting with anti-RD21 and AtSerpin antibodies
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