BINDING OF A SUBSTRATE-ANALOG CAN INDUCE COOPERATIVE STRUCTURE IN THEPLASMIN SERINE-PROTEINASE DOMAIN

Human miniplasminogen and miniplasmin were studied by n.m.r. spectrosc opy and differential scanning calorimetry (d.s.c.) in order to investi gate the structural properties of the serine-proteinase domain. The d. s.c. thermograms of both miniplasminogen and non-inactivated miniplasm in at pH 4.0 can be closely fitted to two transitions, at 62 +/- 2 and 72 +/- 2-degrees-C, corresponding to unfolding of the kringle 5 and p roteinase domains respectively. No evidence was found, under these con ditions, for non-co-operative unfolding of the proteinase domain. On i nactivation of miniplasmin with an affinity label, a number of additio nal resonances arising from residues of the proteinase domain are obse rved in resolved regions of the n.m.r. spectrum. A combination of vari able-temperature n.m.r. and d.s.c. has shown that part of the proteina se domain undergoes a major conformational transition on heating which is distinct from the unfolding of the remainder of the proteinase dom ain or the kringle 5 domain. This additional transition occurs at a te mperature that depends on the nature of the affinity label present and is not observed in the absence of an inactivating agent. These result s provide direct evidence for the existence of a region of the protein ase domain which, under these conditions, becomes structured only in t he presence of a bound substrate.