SECONDARY STRUCTURE AND ZINC LIGATION OF HUMAN RECOMBINANT SHORT-FORMSTROMELYSIN BY MULTIDIMENSIONAL HETERONUCLEAR NMR

Stromelysin-1, a member of the matrix metalloendoprotease family, is a zinc protease involved in the degradation of connective tissue in the extracellular matrix. As a step toward determining the structure of t his protein, multidimensional heteronuclear NMR experiments have been applied to an inhibited truncated form of human stromelysin-1. Extensi ve H-1, C-13, and N-15 sequential assignments have been obtained with a combination of three- and four-dimensional experiments. On the basis of sequential and short-range NOEs and C-13alpha chemical shifts, two helices have been delineated, spanning residues Asp-111 to Val-127 an d Leu-195 to Ser-206. A third helix spanning residues Asp-238 to Gly-2 47 is characterized by sequential NOEs and C-13alpha chemical shifts, but not short-range NOEs. The lack of the latter NOEs suggests that th is helix is either distorted or mobile. Similarly, sequential and inte rstrand NOEs and C-13alpha chemical shifts characterize a four-strande d beta-sheet with three parallel strands (Arg-100 to Ile-101, Ile-142 to Ala-147, Asp-177 to Asp-181) and one antiparallel strand (Ala-165 t o Tyr-168). Two zinc sites have been identifed in stromelysin [Salowe et al. (1992) Biochemistry 31, 4535-4540]. The NMR spectral properties , including chemical shift, pH dependence, and proton coupling of the imidazole nitrogens of six histidine residues (151, 166, 179, 201, 205 , and 211), invariant in the matrix metalloendoprotease family, sugges t that these residues are zinc ligands. NOE data indicate that these h istidines form two clusters: one ligates the catalytic zinc (His-201, -205, and -211), and the other ligates a structural zinc (His-151, -16 6, and -179). Heteronuclear multiple quantum correlated spectra and sp ecific labeling experiments indicate His-151, -179, -201, -205, and -2 11 are in the (N(delta)H)-H-1 tautomer and His-166 is in the (N(epsilo n)H)-H-2 tautomer.