Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into b-sheet A (a1-proteinase inhibitoryPittsburgh variantyserpin covalent complexyf luorescence resonance energy transfer)

To determine the location of the proteinase in the covalent serpin-proteinase complex we prepared seven single-cysteine-containing variants of the Pittsburgh variant of the serpin a1-proteinase inhibitor, and we labeled each cysteine with the dansyl f luorophore. The dansyl probes were used to determine proximity of the proteinase trypsin in covalent and noncovalent complexes with the serpin, both by direct perturbation and by fluorescence energy transfer from tryptophans in trypsin to dansyl. Large direct effects on dansyl f luorophores were seen for only two positions in covalent complex and one position in noncovalent complex. Distances ranging from <14 Å to 64 Å were used to severely constrain possible structures for the complex. The structure consistent with both distance constraints and direct perturbations of the dansyl f luorophores placed the proteinase at the distal end of the serpin from the initial docking site. This position for the proteinase requires complete translocation of the proteinase from one end of the serpin to the other and full insertion of the reactive center loop into b-sheet A to form the kinetically trapped complex. The consequent tight juxtapositioning of serpin and proteinase could explain how distortion of the proteinase active site can occur and hence how many combinations of serpin and proteinase can be inhibited by a common conformational change mechanism. Elucidation of the structure of the serpin-proteinase complex remains the holy grail in trying to understand how serpins inhibit serine proteinases by a kinetic trap mechanism. Serpins inhibit proteinases by a branched pathway, suicide substrate inhibition mechanism (Fig. 1) in which a peptide bond in the exposed reactive center loop is initially recognized as an appropriate proteolytic cleavage site by proteinase, which thereby forms an initial noncovalent Michaelis complex. Formation of this complex is followed by attack by the proteinase active site on the peptide bond (1). Although there may be special cases of particular serpin-proteinase pairs where reaction stops at the Michaelis complex (2), formation of the serpin-proteinase complex under most circumstances involves progression of this initial noncovalent complex to the covalent acyl enzyme intermediate (E-I in Fig. 1), release of the newly formed amino terminus (P19 residue) (3), and insertion of the now unconstrained reactive center loop into b-sheet A. Because the proteinase is covalently linked to the P1 residue of the serpin through an ester linkage, any insertion of the reactive center loop must involve concomitant proteinase translocation. At a point during insertion of the reactive center loop into b-sheet A, a physical interaction is thought to occur between the serpin and the proteinase that is sufficiently large enough to alter the properties of the proteinase (4) and thereby render it catalytically incompetent. The resulting structure represents the kinetically trapped covalent serpin-proteinase complex (E-I†). Several studies support such a general scheme by demonstrating movement of the proteinase as a necessary part of formation of this complex, though without definitively showing where the proteinase is located in the final complex (5–7), and also by providing evidence for structural changes within the proteinase in the final complex (8–11). One study, using a combination of chemical cross-linking and fluorescence energy transfer, concluded that only partial insertion of the reactive center loop into b-sheet A of the serpin occurs and that the proteinase consequently moves only part way down the body of the serpin to a final resting place flanking helix F (Fig. 2, right-hand structure, green trypsin) (6). A recent study from this laboratory was, however, very much at variance with this conclusion (7). Fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD) reporter groups were used to indicate proximity of the proteinase in the complex and strongly implied that the proteinase was not in the vicinity of helix F, but instead at the bottom of the serpin. Although some qualitative fluorescence resonance energy transfer measurements were also attempted, they used nonspecifically localized labels on the proteinase and could therefore not be used to accurately triangulate the position of the proteinase. Because of this remaining fundamental difference in conclusions between these two studies (6, 7) we sought to more definitively determine the location of the proteinase in covalent complex with a serpin. The present report presents the results of this study, using trypsin as the proteinase and the Pittsburgh variant of a1PI (12) as the serpin. MATERIALS AND METHODS Site-Directed Mutagenesis. Site-directed mutagenesis was carried out on a double-stranded pET16b plasmid (Novagen) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: a1PI, a1-proteinase inhibitor; SI, stoichiometry of inhibition; NBD, 7-nitrobenz-2-oxa-1,3-diazole. *To whom reprint requests should be addressed. e-mail: pgettins@ tigger.cc.uic.edu. FIG. 1. Branched pathway mechanism for serpins as suicide substrate inhibitors. E, proteinase; I, serpin; EzI, the noncovalent Michaelis complex; E-I, the covalent acyl enzyme intermediate prior to loop insertion, E-I† the kinetically trapped covalent acyl enzyme intermediate; and I*, cleaved serpin. The rate constants k3 and k4 are for the competing substrate and inhibition pathway and determine the stoichiometry of inhibition (SI) as SI 5 k4y(k3 1 k4).