Canonical Inhibitor-like Interactions Explain Reactivity of α1-Proteinase Inhibitor Pittsburgh and Antithrombin with Proteinases

Abstract

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, ␣ 1 -proteinase inhibitor (␣ 1 PI) Pittsburgh (P1 Met 3 Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound ␣ 1 PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2 . By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with ␣ 1 PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombinthrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for ␣ 1 PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.Although antithrombin is the principal inhibitor of the blood coagulation proteinases thrombin and factor Xa, its rate of reaction with each of these proteinases is slow in the absence of heparin (1, 2). In marked contrast, a variant of the serpin ␣ 1 -proteinase inhibitor that contains a P1 mutation of methionine to arginine, named ␣ 1 PI 1 Pittsburgh, was found to be a very effective inhibitor of thrombin, with a second order rate constant for thrombin inhibition 17-fold higher than that of antithrombin that had been conformationally activated by pentasaccharide heparin (3, 4). In the individual who carried the Pittsburgh mutation, the potent thrombin inhibitory capability of this variant serpin resulted in hemorrhage and ultimately death.In antithrombin, heparin is a multifactorial enhancer that can not only act as a bridging cofactor to bring serpin and proteinase together but can also induce a conformational change in antithrombin that results in expulsion of the two distal residues of the RCL from ␤-sheet A (5, 6). It has been proposed that this loop expulsion permits alteration of the RCL conformation in the vicinity of the scissile bond to a more optimal one for interaction with proteinase and so contributes significantly to the heparin-induced acceleration of proteinase inhibition. In keeping with this proposal, the RCL conformation of antithrombin in published x-ray structures has the P1 arginine residue turned inwards toward the body of the protein, where it forms a salt bridge to Glu 255 (7,8). However, these x-ray structures are all of antithrombin dimers that contain one molecule of functionally active antithrombin and one in an inactive, latent conformation. Importantly, the RCL of the functional molecule forms the interface with the latent molecule, as an extra strand of ␤-sheet C of that molec...

How the Serpin α1-Proteinase Inhibitor Folds

“…From x-ray structures of wild-type ␣ 1 PI it is known that this is a highly solvent-exposed position (16,17). Insertion of the RCL into ␤-sheet A with concomitant longdistance movement of the P9 residue as it becomes part of ␤-sheet A results in a large change in environment for the P9 side chain, as has been found in studies on complex formation with proteinases (18).…”

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

“…Representative gel and blot from three experiments are shown. a hydrophobic groove that imparts broad P2 specificity to trypsin (9,11). In addition, the main chain N of position 104 forms an H bond with position 100 that stabilizes the loop between amino acids 97 and 107.…”

Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

“…Mechanism-based inhibition of proteinase by a serpin. Molecular models of the Michaelis complex (E⅐I, Scheme 1) and the inhibitory complex (E-I*) obtained for ␣1-proteinase inhibitor Pittsburgh and S195A trypsin (E⅐I) (35) and ␣1-proteinase inhibitor and porcine pancreatic elastase (E-I*) (34). The serpin molecule is shown in yellow; the RCL is shown in pink, and ␣HF is colored in cyan.…”

“…1) (32)(33)(34). Despite the fact that the structures of both the Michaelis complex (Scheme 1, E⅐I) (35) and the final inhibitory complex (E-I*) (34,36) are known for several serpins and proteinases, it is not clear what transient intermediates form on the reaction pathway from E⅐I to E-I*, as well as how bifurcation is regulated between the inhibitory and substrate pathways of the serpin reaction (Scheme 1) (31).…”