Protein engineering is the process of modifying proteins with the goal of obtaining a protein that is more suitable for a particular application than the unmodified version. Protein engineering of proteases has mainly focused on modifying their specificity and improving enzyme activity, while engineering of proteases for the purpose of oligomerization has remained unexplored.
The aim of this master thesis was to identify potential protein-protein interface residues and to design a novel homodimeric state of the papain-like cysteine peptidase cathepsin K. It is highly expressed in osteoclasts as the major collagenolytic protease in bone turnover. Since its excessive activity is associated with various pathological conditions, its structure, function, and enzyme activity have been studied in detail. Therefore, cathepsin K is a suitable target for protein engineering of oligomeric states of the enzyme that would be more stable and active in comparison to monomeric state of cathepsin K.
To this end, using bioinformatics tools to predict protein-protein interface residues and molecular docking, we predicted three different possibilities for homodimerization of cathepsin K. The best computer-evaluated structure is a symmetric homodimer calculated with the program SymmDock with surface interaction residues Lys9, Pro15, Gly168 and Ile179. Its in vitro formation and stability would need to be evaluated experimentally.
Furthermore, we produced two recombinant mutant variants of cathepsin K with insertion sequences responsible for the dimerization of cathepsin X. The mutant versions of cathepsin K were inactive, from which we concluded that the insertion sequences significantly affect protein folding or prodomain - catalytic domain interactions and consequent zymogen activation. By size-exclusion chromatography, we confirmed that one of the mutants exists only in the monomeric state despite the introduction of dimerization loops. The mutant with only one dimerization loop could potentially form a dimer, but its structure would need to be further investigated.