The N-terminus of collagenase MMP-8 determines superactivity and inhibition: A relation of structure and function analyzed by biomolecular interaction analysis

Tissue inhibitors of metalloproteinases (TIMPs) are the physiological, spec ific inhibitors of matrix metalloproteinases (MMPs) forming tight, noncoval ent complexes. Therefore they control the proteolytic activity of MMPs towa rd the extracellular matrix. To analyze the inhibition of the "activated" a nd "superactivated" variants of human neutrophil collagenase (MMP-8) by TIM P-2, we determined complex dissociation constants using biomolecular intera ction analysis (BIA). As it is known that the association rate constants ca n exceed the limits of the BIA instruments, the biomolecular interaction an alysis was used to examine the equlibrium situation. The dissociation const ants were determined by fitting the parameters of the mathematical term for the binding of collagenase onto the TIMP-coupled sensor chip surface to th e saturation curve derived from individual sensorgrams. The resulting value s are in the nanomolar range and correlate with the results of fluorescence kinetics. These data reveal that TIMP-2 (the recombinant inhibitory domain of human TIMP-2 and bovine TIMP-2 isolated from seminal plama) is a better inhibitor of the activated neutrophil collagenase than of the superactivat ed variant (the recombinant catalytic domain of human MMP-8). It has been d emonstrated by X-ray analysis that the N-terminal heptapeptide only of supe ractivated MMP-8 is attached by a salt bridge and hydrophobic interaction t o the C-terminal helix. Because these interactions have to be disrupted in the complex formation with TIMP we assume that the activated variant enable s higher flexibility and a tighter induced fit in the complex formation. Th erefore superactivation of MMP-8 correlates with weaker inhibition by TIMP- 2.