Structural characterisation 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 proteinases. The 1H-NMR and 15N-NMR resonances of human stefin A have been sequentially assigned using two-dimensional homonuclear and heteronuclear NMR techniques in conjunction with three-dimensional heteronuclear methods. Characteristic sequential and medium range NOE contacts, J constants and hydrogen exchange data have been used to identify the secondary structural elements of the protein which consists of five anti-parallel beta-strands and a single alpha-helix. There is much similarity 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 regions which are fundamental to the binding event. The principal difference 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, these regions are the five N-terminal residues and the second binding loop, which form a turn and a short helix respectively, in the bound conformation of stefin B.