Arabidopsis AtSerpin1, Crystal Structure and in Vivo Interaction with Its Target Protease RESPONSIVE TO DESICCATION-21 (RD21)

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 Å . 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 fulllength 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.Protease cascades are prominent mediators of rapid physiological responses in animals, playing a role in cellular immunity, blood clotting, and development. The proteolytic specificity of the serine and cysteine proteases involved dictates the fidelity of these reactions. The serpins are an important group of proteins that curb the activity of these cascades through specific irreversible inhibition of the proteases. For example, in Drosophila, the necrotic (nec) gene encodes a protease inhibitor of the serpin family. Necrotic protein controls a proteolytic cascade that activates the innate immune response to fungal and Gram-positive bacterial infections (1). In nec null mutants, Toll-mediated immune responses are constitutively activated, even in the absence of infection, implying that Nec continually restrains this immune response. As opposed to other types of protease inhibitors, serpins offer both an irreversible and tunable type of inhibition (reviewed in Ref.2). In their native conformation, serpins are in a stressed (spring-loaded) state with a solvent-exposed reactive center loop (RCL).3 Specific residues of the RCL are precisely accommodated by the target protease active site. Upon cleavage of the serpin peptide bond linking the P1 and ...

Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana

“…S4). Similar results have been described by Lampl et al (13) and were shown to be a result of cleavage of AtSerpin1 in the RCL. When cysteine protease inhibitors such as E-64, leupeptin, and antipain were coinfiltrated with DCG-04, neither the upper nor the lower AtSerpin1 bands appeared.…”

“…When the seedlings begin to de-etiolate, RD21 is released from the complex to counteract digestive proteases in the arthropod gut, conferring efficient protection (16). In greening and mature seedlings, RD21 is operative as a prodeath factor that triggers programmed host cell death once released from the vacuole (13,14). Binding of RD21 to AtSerpin1, however, is a mechanism to control RD21's activity as prodeath factor (13,14).…”

“…In greening and mature seedlings, RD21 is operative as a prodeath factor that triggers programmed host cell death once released from the vacuole (13,14). Binding of RD21 to AtSerpin1, however, is a mechanism to control RD21's activity as prodeath factor (13,14). Changes in vacuole permeability and protease compartmentalization are supposed to account for this set-point control mechanism (8,14).…”

“…RD21 accumulates in ER bodies and lytic vacuoles; some studies revealed accumulation in cell walls/apoplastic spaces (12). Evidence suggests the activity of RD21 is regulated by at least two different protease inhibitors (PIs): a serine protease inhibitor (serpin)-like activity (13,14) and a Kunitz protease inhibitor-type activity (15)(16)(17). Serpins have been found in animals and plants and feature a reactive center loop (RCL), which displays a protease target sequence (18).…”

“…Cleavage of the RCL results in an irreversible, covalent serpin::protease adduct (19)(20)(21). Similarly, Arabidopsis Serpin1 forms RD21::Serpin1 adduct (henceforth referred to as RD21-Serpin1) in response to fungal pathogens and was suggested to provide some set-point control of RD21 activity during programmed cell death (8,11,13,14).…”