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 1HN, 15N, 13C alpha, 13CO, and 1H alpha NMR assignments for the catalytic domain of human fibroblast collagenase (HFC). Three independent assignment pathways (matching 1H, 13C alpha, and 13CO resonances) were used to establish sequential connections. The connections using 13C alpha resonances were obtained from HNCOCA and HNCA experiments; 13CO connections were obtained from HNCO and HNCACO experiments. The sequential proton assignment pathway was established from a 3D (1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and 15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement with recent X-ray structures of human fibroblast collagenase and human neutrophil collagenase as well as similar to recent NMR structures of a highly homologous protein, stromelysin. All three helices were located; a five-stranded beta-sheet (four parallel strands, one antiparallel strand) was also determined. beta-Sheet regions were identified by cross-strand d alpha N and d NN connections and by strong intraresidue d alpha N correlations, and were corroborated by observing slow amide proton exchange. Chemical shift changes in a selectively 15N-labeled sample suggest that substantial structural changes occur in the active site cleft on the binding of an inhibitor.
McCoy
M A
MA
Sterling Winthrop Pharmaceuticals Research Division, Collegeville, PA 19426, USA.
Dellwo
M J
MJ
Schneider
D M
DM
Banks
T M
TM
Falvo
J
J
Vavra
K J
KJ
Mathiowetz
A M
AM
Qoronfleh
M W
MW
Ciccarelli
R
R
Cook
E R
ER
Pulvino
T A
TA
Wahl
R C
RC
Wang
H
H
D000595:Amino Acid Sequence; D017364:Collagenases / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017421:Sequence Analysis
D017364:Collagenases