Conformational variability of matrix metalloproteinases: Beyond a single 3D structure

doi:10.1073/pnas.0407106102

Conformational variability of matrix metalloproteinases: Beyond a single 3D structure

^*Magnetic Resonance Center (Centro di Risonanze Magnetiche), University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy; ^†Department of Chemistry, University of Florence, Via Della Lastruccia 5, 50019 Sesto Fiorentino, Italy; ^§Department of Chemistry, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy; and ^¶Department of Agricultural Biotechnology, University of Florence, Piazzale le delle Cascine 24, 50144 Florence, Italy

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved March 7, 2005 (received for review September 24, 2004)

Abstract

The structures of the catalytic domain of matrix metalloproteinase 12 in the presence of acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid have been solved by x-ray diffraction in the crystalline state at 1.0 and 1.3-Å resolution, respectively, and compared with the previously published x-ray structure at 1.2-Å resolution of the adduct with batimastat. The structure of the N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid adduct has been solved by NMR in solution. The three x-ray structures and the solution structure are similar but not identical to one another, the differences being sizably higher in the loops. We propose that many of the loops show a dynamical behavior in solution on a variety of time scales. Different conformations of some flexible regions of the protein can be observed as “frozen” in different crystalline environments. The mobility in solution studied by NMR reveals conformational equilibria in accessible time scales, i.e., from 10^–5 s to ms and more. Averaging of some residual dipolar couplings is consistent with further motions down to 10^–9 s. Finally, local thermal motions of each frozen conformation in the crystalline state at 100 K correlate well with local motions on the picosecond time scale. Flexibility/conformational heterogeneity in crucial parts of the catalytic domain is a rule rather than an exception in matrix metalloproteinases, and its extent may be underestimated by inspection of one x-ray structure. Backbone flexibility may play a role in the difficulties encountered in the design of selective inhibitors, whereas it may be a requisite for substrate binding and broad substrate specificity.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: bertini{at}cerm.unifi.it.
Author contributions: I.B. designed research; V.C., M.C., M.F., Y.-M.L., B.T., and P.T. performed research; V.C., M.F., Y.-M.L., C.L., S.M., and P.T. analyzed data; and I.B., C.L., and S.M. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: MMP, matrix metalloproteinase; NNGH, N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid; AHA, acetohydroxamic acid; BB, backbone; NOE, nuclear Overhauser effect; RDC, residual dipolar coupling; RMSD, RMS deviation.
Data deposition: The atomic coordinates and structure factors for the x-ray crystal structures of the AHA and NNGH adducts have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1Y93 and 1RMZ, respectively). The solution structure of the NNGH and the list of upper experimental constraints used for structure calculations have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1YCM and 1Z3J). The NMR chemical shift values have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession no. 6444).