ASSIGNMENTS AND STRUCTURE DETERMINATION OF THE CATALYTIC DOMAIN OF HUMAN FIBROBLAST COLLAGENASE USING 3D DOUBLE AND TRIPLE-RESONANCE NMR-SPECTROSCOPY

We report here the backbone (HN)-H-1, N-15, C-13(alpha), (CO)-C-13, an d H-1(alpha) NMR assignments for the catalytic domain of human fibrobl ast collagenase (HFC). Three independent assignment pathways (matching H-1, C-13(alpha), and (CO)-C-13 resonances) were used to establish se quential connections. The connections using C-13(alpha) resonances wer e obtained from HNCOCA and HNCA experiments; (CO)-C-13 connections wer e obtained from HNCO and HNCACO experiments. The sequential proton ass ignment pathway was established from a 3D (H-1/N-15) NOESY-HSQC experi ment. Amino acid typing was accomplished using C-13 and N-15 chemical shifts, specific labeling of N-15-Leu, and spin pattern recognition fr om DQF-COSY. The secondary structure was determined by analyzing the 3 D (H-1/N-15) NOESY-HSQC. A preliminary NMR structure calculation of HF C was found to be in agreement with recent X-ray structures of human f ibroblast collagenase and human neutrophil collagenase as well as simi lar to recent NMR structures of a highly homologous protein, stromelys in. All three helices were located; a five-stranded beta-sheet (four p arallel strands, one antiparallel strand) was also determined. beta-Sh eet regions were identified by cross-strand d(alpha N) and d(N,N) conn ections and by strong intraresidue d(alpha N) correlations, and were c orroborated by observing slow amide proton exchange. Chemical shift ch anges in a selectively N-15-labeled sample suggest that substantial st ructural changes occur in the active site cleft on the binding of an i nhibitor.