AbstractCollagenolysis, the catabolism of triple helical collagen, is essential for the physiological remodeling of connective tissues during growth and development. Aberrant collagen degradation is a feature of both inflammatory diseases and cancer cell invasion. Matrix metalloproteinase-1 (MMP-1) is a known collagenase and previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilising collagenin preparation for hydrolysis by the catalytic (CAT) domain. More recently,conformational freedom and domain separation of the CAT and HPX domains has been proposed to play a role in collagen degradation. This study aims to explore HPX mediated collagen recognition and the postulated flexible state of MMP-1, in order to enhance our understanding of the collagenolytic mechanism.
Here, biophysical methods have been used to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. A programme of site-directed mutagenesis was used to produce an extensive library of recombinant proteins. Surface plasmon resonance (SPR) has been used to characterise a previously unknown collagen binding site (exosite) located in blade 1 of the HPX domain and small angle x-ray scattering has been used to probe the conformational freedom and transient domain separation in mutant forms of both zymogen and mature MMP-1enzymes.
Significant reductions in THP affinity were observed on mutation of eitherPhe301, Val319 and Asp338, residues forming part of a “ball and socket” joint in the CAT-HPX interface. Disruption of the CAT-HPX interface by mutagenesis, of any of these three residues, severely impacts collagen recognition. Conversely, the reduced collagen binding activity of a "stapled" mutant (in which the CAT and HPX domains are constrained with a disulphide bridge) indicates that the ability of the domains to transiently dislocate is also important. Thus, a balanced equilibrium between these compact and dislocated states is an essential feature of MMP-1 collagenolytic activity.
|Date of Award||Aug 2013|
|Supervisor||Andrew Pickford (Supervisor) & Geoff Kneale (Supervisor)|