Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase

Fibroblast collagenase (MMP-1), a 169-residue protein with a molecular mass of 18.7 kDa, is a matrix metalloproteinase which has been associated with pathologies such as arthritis and cancer. The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances, determination of the secondary structure and analysis of ¹⁵N relaxation data of the inhibitor-free catalytic fragment of recombinant human fibroblast collagenase (MMP-1) are presented. It is shown that MMP-1 is composed of a β-sheet consisting of five β-strands in a mixed parallel and antiparallel arrangement (residues 13-19, 48-53, 59-65, 82-85 and 94-99) and three α-helices (residues 27-43, 112-124 and 150-160). This is nearly identical to the secondary structure determined from the refined X-ray crystal structures of inhibited MMP-1. The major difference observed between the NMR solution structure of inhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is the dynamics of the active site. The 2D ¹⁵N-¹H HSQC spectra, the lack of information in the ¹⁵N-edited NOESY spectra, and the generalized order parameters (S²) determined from ¹⁵N T₁, T₂ and NOE data suggest a slow conformational exchange for residues comprising the active site (helix B, zinc ligated histidines and the nearby loop region) and a high mobility for residues Pro¹³⁸-Gly¹⁴⁴ in the vicinity of the active site for inhibitor-free collagenase. In contrast to the X-ray structures, only the slow conformational exchange is lost in the presence of an inhibitor.