The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites.

BACKGROUND: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin-protease complex have been proposed. In the absence of a spatial structure of a serpin-protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial. RESULTS: We used a recently developed method for making precise distance measurements, based on donor-donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1') were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into beta sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease-inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker. CONCLUSIONS: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

Ovulation in plasminogen-deficient mice.

Many different studies suggest that plasmin generated from plasminogen plays a crucial role in the degradation of the follicular wall at the time of ovulation. We have assessed the physiological relevance of plasmin on ovulation by studying plasminogen-deficient mice. Ovulation efficiency (mean number of ova released per mouse) was determined both in a standardized ovulation model in which 25-day-old immature mice were injected with finite amounts of gonadotropins to induce ovulation and during physiological ovulation using adult normally cycling mice. Our results revealed that the temporal onset of follicular wall rupture (first ova observed in bursa or oviduct) was not delayed in plasminogen-deficient mice during gonadotropin-induced ovulation. However, there was a trend toward slightly reduced ovulation efficiency in the plasminogen-deficient mice. This reduction was only 13% and not statistically significant (P = 0.084) and may be connected to a delayed maturation of these mice manifested in reduced body and ovary weights. During physiological ovulation adult plasminogen-deficient mice had normal ovulation efficiency compared with plasminogen wild-type mice. Taken together our results indicate that under the conditions used in this study plasmin is not required for efficient follicular rupture or for activation of other proteases involved in this process. Alternatively, the role of plasmin may be effectively compensated for by other mechanisms in the absence of plasmin.

The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.

The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor-donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C(alpha)-atoms of the positions V106C-H185C, H185C-M266C, and M266C-V106C were 60.9, 30.8, and 55.1 A, respectively. These are in good agreement with the distances of 54 +/- 4, 38 +/- 3, and 55 +/- 3 A, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C(alpha)-atoms physically cannot coincide, there is a reasonable agreement between the methods.