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Characterisation of the collagen binding domain of gelatinase A : involvement of specific residues in the fibronectin type II modules in substrate recognition

University of British Columbia

The matrix metalloproteinase (MMP) family of endopeptidases can collectively degrade many components of the extracellular matrix. Their proteolytic activities have been implicated in normal processes such as extracellular matrix turnover and certain pathological conditions such as periodontitis, arthritis, and tumour metastasis. Based upon domain composition gelatinase A is separated from the other MMPs by the insertion of three contiguous fibronectin type II modules into its catalytic domain. Since it was determined that a recombinant domain consisting of the three fibronectin type II modules bound to native type I collagen this domain was termed the collagen binding domain (CBD). The function of the CBD is to donate substrate binding exosites to allow for broader enzyme specificity. Considering that the CBD contains exosites for binding native/denatured collagen, this domain was subjected to site-directed mutagenic studies to elucidate essential residues involved in collagen binding. The binding properties of the collagen binding domain to denatured type I collagen (gelatin) was investigated using a recombinant protein constructed of the second and third fibronectin type II module (rCBD23). Ten mutations were performed within the rCBD23 protein. Since the binding of substrate to the CBD is via a hydrophobic pocket, the mutations F264A, F264Y, F266A, F266Y, F322A, F324A, F322Y, F324Y, F264A/F322A, and F266A/F324A were introduced into rCBD23 in order to determine the effect of removing hydrophobic character. It was found that there was a decrease in the binding of the mutant proteins that was proportional to the distance between the mutated residue side chain and a strictly conserved tryptophan at the base of the hydrophobic pocket. Complete abrogation of gelatin binding was observed in the double mutant F266A/F324A. The observed effect was proposed to be the result of destabilisation of the strictly conserved tryptophan that forms the base of the hydrophobic pocket. These studies have furthered our knowledge and understanding of the interactions of CBD with substrate. The present study, combined with results from other studies, could be used to synthesize compounds that are specific to the CBD and are able to irreversibly inhibit the binding of CBD to substrate. A drug that would preferentially block binding of the CBD to collagen is proposed to reduced the metastasis of tumour cells by abolishment of type IV collagen binding. The overall effect is decreased tumour migration from the primary site of development and therefore substantially decreasing tumour progression.

Thesis/Dissertation

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2009-08-05

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http://hdl.handle.net/2429/11743

Dental Science

University of British Columbia

UBCV

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