W0390

Non-covalent α₁-Proteinase Inhibitor-Trypsin Complex: Serpins as Canonical Inhibitors of Proteinases. Alexey A. Dementiev¹, Miljan Simonovic¹, Peter G.W. Gettins¹ and Karl Volz², ¹Dept. of Biochemistry & Molecular Biology, and ²Dept. of Microbiology and Immunology, Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612.

Serpins are the predominant endo-proteinase inhibitors in mammals and are involved in diverse physiological processes such as coagulation, fibrinolysis, angiogenesis, and complement activation. The standard model of serpin inhibition is based on complementary binding of the inhibitor to the active site of the target proteinase. After reacting with the proteinase as a substrate and forming a covalent acyl-enzyme intermediate, the serpin undergoes a massive conformational change that results in kinetic trapping of the destabilized proteinase.

The serpin known as α₁-Proteinase inhibitor (α₁-PI) has neutrophil elastase as its normal target. However, one naturally occurring variant (known as Pittsburgh, or α₁-PI_Pitt) has a methionine to arginine mutation at the P1 site that changes its specificity to thrombin, which results in a fatal bleeding disorder. In an attempt to understand the structural basis for this change in serpin specificity, we determined the crystal structures of α₁-PI_Pitt in both the free and S195A trypsin-bound form. Kinetic analyses of the inhibition rates of trypsin and thrombin by α₁-PI_Pitt and other variants were also performed.

Data collection at the SER-CAT beamline of APS yielded 2.6 Å and 2.3 Å resolution data from crystals of α₁-PI_Pitt, free and in complex with trypsin, respectively. Both structures have been solved and refined to an R_free of 23%. Their structures and kinetic data suggest that this and other Michaelis-like serpins-proteinase complexes resemble classical proteinase inhibitor-proteinase complexes. This finding of canonical binding argues that in general, serpins’ mechanisms of Michaelis complex formation are closer to those of the more traditional lock-and-key type inhibitors than previously thought.