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 f luorescence resonance energy transfer to measure the separation between f luorescein attached to a single cysteine on the serpin and tetramethylrhodamine conjugated to the proteinase. The interf luorophore separation was determined for the noncovalent Michaelis-like complex formed between ␣ 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 f luorophores of Ϸ21 Å 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 interf luorophore 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 ␤-sheet A of the serpin.

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

“…No contacts were present between the serpin body and the proteinase, unlike the noncovalent complex of another serpin, heparin cofactor II, with S195A thrombin. Surprisingly, considering the high stability of the ␣ 1 PI Pittsburgh-S195A trypsin complex (K d ϳ5 nM) (15), contacts between the serpin RCL and the proteinase are quite limited (Fig. 1c).…”

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

“…and is such that it is dependent on 2 , the orientation factor, which is routinely given a value of 2/3 indicating that both donor and acceptor fluorophores exhibit isotropic motion (47)(48)(49), n the experimentally determined refractive index of 1.3364 (using a Carl Zeiss ABBE-type refractometer), D the experimental quantum yield of the donor fluorophore, and J DA the overlap integral for the donor and acceptor fluorophore pair, which was previously determined to be 3.06 ϫ 10 Ϫ13 M Ϫ1 cm 3 (49). The quantum yield was measured by comparison of the emission spectrum of PAI-1 P1Ј-FL to that of a fluorescein reference in 0.1 M NaOH ( ϭ 0.95) and evaluated as 0.143 by Equation 5,…”

“…A calculated R o of 45 Å for this donor and acceptor pair was determined from Equation 4 using an experimentally determined quantum yield of 0.143 for the PAI-1 P1Ј-FL donor (Equation 5). The standard assumption that both the donor and acceptor probes exhibited isotropic motion was invoked, and therefore a value of 2/3 was valid for the orientation factor 2 (47)(48)(49). This value, in combination with the above determination for the efficiency of FRET, was used to calculate the intermolecular distance between the fluorescence labels on PAI-1 P1Ј-FL and PAI-1 P1Ј-TMR when in a complex with monomeric vitronectin by evaluation of Equation 3, yielding a calculated distance of 57 Å.…”

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

“…complex has not yet been conclusively shown by X-ray crystallography, recent studies using fluorescence resonance energy transfer (FRET) (Stratikos & Gettins 1997;Wilczynska et al 1997) are helping to elucidate both the catalytic mechanism(s) responsible for the range of structural states found within the serpin superfamily, a how the structure/function relationships of these proteins have evolved.…”

“…Using fluorescence resonance energy transfer (FRET), two recent studies measured intra-and inter-molecular distances between various residues in two serpin-protease complexes (uPA:PAI-l and trypsin:a,-PI). Stratikos & Gettins (1997) measured an increase of ~2lA in the distance between fluorophores attached to specific residues in trypsin and oc r PI upon formation of the stable covalent complex, compared to the initial Michaelis-like complex. Wilczynska et al (1997), using a similar technique, measured a separation of ~60A between the P3 and P1' residues of PAI-1 upon formation of a stable covalent complex with uPA.…”