Major proteinase movement upon stable serpin-proteinase complex formation

Abstract

To determine whether formation of the stable complex between a serpin and a target proteinase involves a major translocation of the proteinase from its initial position in the noncovalent Michaelis complex, we have used fluorescence resonance energy transfer to measure the separation between fluorescein attached to a single cysteine on the serpin and tetramethylrhodamine conjugated to the proteinase. The interfluorophore separation was determined for the noncovalent Michaelis-like complex formed between alpha 1-proteinase inhibitor (Pittsburgh variant) and anhydrotrypsin and for the stable complex between the same serpin and trypsin. A difference in separation between the two fluorophores of approximately 21 A was found for the two types of complex. This demonstrates a major movement of the proteinase in going from the initial noncovalent encounter complex to the kinetically stable complex. The change in interfluorophore separation is most readily understood in terms of movement of the proteinase from the reactive center end of the serpin toward the distal end, as the covalently attached reactive center loop inserts into beta-sheet A of the serpin.

Figure 1

The branched suicide substrate pathway of serpins (I) interacting with proteinase (E), showing the possible types of structure for the different intermediates and products. The initial noncovalent Michaelis-like complex (EI) is expected to resemble non-serpin proteinase–inhibitor complexes such as those of bovine pancreatic trypsin inhibitor (BPTI) and trypsin. The structure of cleaved serpin (I*; the product of the substrate branch of the pathway) is known for several serpins and has the cleaved reactive center loop completely inserted into β-sheet A. Two different types of structure are shown for the stable complex (EI⁺). In one, little or no movement of the proteinase has occurred and stabilization results largely from noncovalent interaction with the serpin. In the other, the P1–P1′ peptide bond has been cleaved, permitting complete loop insertion, with the intermediate trapped at the stage of the covalent acyl enzyme intermediate. The EI⁺ complex can decay very slowly (k₅) to cleaved serpin and free proteinase.

Figure 2

Ability of inactivated (anhydro-) trypsin to form tight complex with α₁-proteinase inhibitor Pittsburgh but not with wild-type α₁-proteinase inhibitor. Titration of α₁-proteinase inhibitor Pittsburgh (○) into a solution of anhydrotrypsin (4.5 μM) in the presence of p-aminobenzamidine (100 μM) resulted in stoichiometric displacement of the noncovalently bound fluorescent probe, indicating tight 1:1 noncovalent complex formation. The solid straight lines are for visual aid only. Similar titration of wild-type α₁-proteinase inhibitor (□) under the same conditions led to negligible complex formation and consequently little change in probe fluorescence.

Figure 4

Quantitiation of the efficiency of fluorescence resonance energy transfer in covalent and noncovalent α₁-proteinase inhibitor Pittsburgh–trypsin complexes. (A) Decrease in fluorescein emission of fluorescein–α₁-proteinase inhibitor Pittsburgh (80 nM) as a function of added tetramethylrhodamine–anhydrotrypsin. The solid line represents the nonlinear least-squares fit of the data to a simple binding equation and gives K_D of ≈5 nM. (B) Time course of decay in fluorescein emission intensity of fluorescein–α₁-proteinase inhibitor Pittsburgh after mixing with two equivalents of tetramethylrhodamine-trypsin in the presence of 10 mM benzamidine to slow the reaction. The solid line is a fit of the data to a bimolecular reaction and gave a second-order rate constant, when corrected for the competitive inhibitor benzamidine, of 1.5 × 10⁷ M⁻¹·sec⁻¹.

Figure 3

Differential efficiencies of fluorescence resonance energy transfer between fluorescein and tetramethylrhodamine in covalent and noncovalent serpin–proteinase complexes. (A) Effect of formation of non-covalent complex between fluorescein–α₁-proteinase inhibitor Pittsburgh and tetramethylrhodamine–anhydrotrypsin. Solid line, fluorescein–α₁-proteinase inhibitor Pittsburgh (82 nM) plus 250 nM unlabeled anhydrotrypsin; dashed line, tetramethylrhodamine–anhydrotrypsin; and dotted line, mixture of fluorescein–α₁-proteinase inhibitor Pittsburgh (82 nM) and saturating levels (250 nM) of tetramethylrhodamine–anhydrotrypsin, showing reduction in fluorescein emission at 517 nm and increase in tetramethylrhodamine fluorescence at 575 nm. (B) Effect of formation of covalent complex between fluorescein–α₁-proteinase inhibitor Pittsburgh and tetramethylrhodamine–trypsin. Solid line, fluorescein–α₁-proteinase inhibitor Pittsburgh (82 nM) plus one equivalent unlabeled trypsin; dashed line, 82 nM tetramethylrhodamine–trypsin; and dotted line, 1:1 mixture of fluorescein–α₁-proteinase inhibitor Pittsburgh and tetramethylrhodamine–trypsin, showing smaller reduction in fluorescein emission and smaller increase in tetramethylrhodamine emission at 575 nm in the complex.

Figure 5

Models of noncovalent and covalent serpin–proteinase complexes. The serpin (solid ribbon) is shown in the center in an orientation such that the reactive center loop in the unreacted molecule is at the top of the molecule and β-sheet A is seen edge-on, running from top to bottom. The F helix is on the surface of β-sheet A at the bottom right side (distal end of β-sheet from the reactive center). The serpin shown is antithrombin in its crystal structure conformation that has an appropriate exposed and extended reactive center loop for initial noncovalent docking with proteinase. However, the structural homology between serpins is such that the location indicated for the Cys-232 residue is equivalent to that in α₁-proteinase inhibitor. The proteinase (three-stranded ribbon) is shown in three different locations corresponding to three distinct types of complex. Two circles are drawn, centered on the position of Cys-232 in α₁-proteinase inhibitor Pittsburgh, with radii of 35 Å and 56 Å, corresponding to the interfluorophore separations measured for the noncovalent and covalent serpin–proteinase complexes, respectively. Structure A (proteinase at top) represents the noncovalent complex of the anhydroproteinase or of the active proteinase in the initial Michaelis-like complex. Structure B (proteinase in the middle) represents a covalent partially loop-inserted (up to P9) covalent complex. Note the abutment of the proteinase against the F α-helix. Structure C (proteinase at the bottom), represents a covalent complex in which the reactive center loop has completely inserted into β-sheet A and has resulted in translocation of the proteinase from the proximal (top) end of the serpin to the distal (bottom) end.