STRUCTURAL CHARACTERIZATION OF HUMAN STEFIN-A IN SOLUTION AND IMPLICATIONS FOR BINDING TO CYSTEINE PROTEINASES

Stefin A is a member of the cystatin superfamily of proteins which are tight and reversibly binding inhibitors of the papain-like cysteine p roteinases. The H-1-NMR and N-15-NMR resonances of human stefin A have been sequentially assigned using two-dimensional homonuclear and hete ronuclear NMR techniques in conjunction with three-dimensional heteron uclear methods. Characteristic sequential and medium range NOE contact s, J constants and hydrogen exchange data have been used to identify t he secondary structural elements of the protein which consists of five anti-parallel beta-strands and a single alpha-helix. There is much si milarity between the secondary structural features of stefin A and the homologous protein stefin B in its complex with papain [Stubbs, M. T. , Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B. & Turk, V. (1990) EMBO. J. 9, 1939-1947] but also some important differences in r egions which are fundamental to the binding event. The principal diffe rence is the presence of two conformationally unrestricted regions in stefin A that form two of the components of the tripartite wedge which docks into the active site of the target proteinase. Specifically, th ese regions are the five N-terminal residues and the second binding lo op, which form a turn and a short helix respectively, in the bound con formation of stefin B.