Conversion of the Kunitz-type module of collagen VI into a highly active trypsin inhibitor by site-directed mutagenesis.

The recombinant Kunitz protease inhibitor module (domain C5) of human collagen alpha 3(VI) chain was previously shown to lack inhibitory activity for proteases with trypsin-like specificity and some other proteases. We have now prepared mutants in the binding loop region including the P1' site (D2889-->A), the P2' site (F2890-->R) and the P3 site (T2886-->P) and in a more remote region (W2907-->V) either as individual substitutions or combinations of them. These mutants were analyzed for their kinetics of binding to trypsin by surface plasmon resonance and for their capacity to inhibit various proteases. Single substitutions (D-->A, T-->P, W-->V) showed an effect only for D->A which bound to trypsin with Kd = 0.25 microM. A 25-100-fold increase in affinity was observed for the double mutants T-->P/D-->A and F-->R/D-->A and approached the affinity of aprotinin (Kd approximately 0.01 nM) in two different triple mutants. These affinities correlated well with the inhibitory capacities of the mutants for trypsin in the cleavage of a large protein and a small peptide substrate. A similar but not completely identical improvement in inhibitory capacity was also observed for leucocyte elastase but not for thrombin. These data could be interpreted in terms of steric interferences or lack of hydrogen bonding of a few critical residues based on three-dimensional structures available for the C5 domain.