STRUCTURAL BASIS FOR DIFFERENT INHIBITORY SPECIFICITIES OF HUMAN CYSTATIN-C AND CYSTATIN-D

Human cystatins C and D share almost identical primary structures of t wo out of the three segments proposed to be of importance for enzyme i nteractions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L. and S. binding region s of the inhibitors are responsible for the different inhibition profi les, and thereby confer biological selectivity, two hybrid cystatins w ere produced in Escherichia coli expression systems. In one hybrid, th e N-terminal segment of cystatin C was placed on the framework of cyst atin D, and the second was engineered with the N-terminal segment of c ystatin D on the cystatin C scaffold. Truncated cystatin C and D varia nts, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin functio n and specificity. The affinities of the four cystatin variants for ca thepsins B, H, L, and S were measured. By comparison with correspondin g results for wild-type cystatins C and D, it was concluded (1) that b oth the N-terminal and framework part of the molecules significantly c ontribute to the observed differences in inhibitory activities of cyst atins C and D and (2) that the N-terminal segment of cystatin C increa ses the inhibitory activity of cystatin D against cathepsin S and cath epsin L but results in decreased activity against cathepsin H. These d ifferences in specificity were explained by the residues interacting w ith the S? subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, nt results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences i n the framework parts, as well as in the N-terminal segments, are crit ical for both inhibitory specificity and potency. Homology modeling wa s used to identify residues likely responsible for the gnerally reduce d inhgiitory potency of cystatin D.