TY - JOUR
T1 - Solution Structure of Inhibitor-Free Human Metalloelastase (MMP-12) Indicates an Internal Conformational Adjustment
AU - Bhaskaran, Rajagopalan
AU - Palmier, Mark O.
AU - Bagegni, Nusayba A.
AU - Liang, Xiangyang
AU - Van Doren, Steven R.
N1 - Funding Information:
This work was supported by the American Heart Association (0455885Z and 0256134Z) and the National Institutes of Health (R01 GM57289). The Inova 600 MHz NMR system was purchased with funding from NSF DBI-0070359 and the University of Missouri.
PY - 2007/12/14
Y1 - 2007/12/14
N2 - Macrophage metalloelastase or matrix metalloproteinase-12 (MMP-12) appears to exacerbate atherosclerosis, emphysema, aortic aneurysm, rheumatoid arthritis, and inflammatory bowel disease. An inactivating E219A mutation, validated by crystallography and NMR spectra, prevents autolysis of MMP-12 and allows us to determine its NMR structure without an inhibitor. The structural ensemble of the catalytic domain without an inhibitor is based on 2813 nuclear Overhauser effects (NOEs) and has an average RMSD to the mean structure of 0.25 Å for the backbone and 0.61 Å for all heavy atoms for residues Trp109-Gly263. Compared to crystal structures of MMP-12, helix B (hB) at the active site is unexpectedly more deeply recessed under the β-sheet. This opens a pocket between hB and β-strand IV in the active-site cleft. Both hB and an internal cavity are shifted toward β-strand I, β-strand III, and helix A on the back side of the protease. About 25 internal NOE contacts distinguish the inhibitor-free solution structure and indicate hB's greater depth and proximity to the sheet and helix A. Line broadening and multiplicity of amide proton NMR peaks from hB are consistent with hB undergoing a slow conformational exchange among subtly different environments. Inhibitor-binding-induced perturbations of the NMR spectra of MMP-1 and MMP-3 map to similar locations across MMP-12 and encompass the internal conformational adjustments. Evolutionary trace analysis suggests a functionally important network of residues that encompasses most of the locations adjusting in conformation, including 18 residues with NOE contacts unique to inhibitor-free MMP-12. The conformational change, sequence analysis, and inhibitor perturbations of NMR spectra agree on the network they identify between structural scaffold and the active site of MMPs.
AB - Macrophage metalloelastase or matrix metalloproteinase-12 (MMP-12) appears to exacerbate atherosclerosis, emphysema, aortic aneurysm, rheumatoid arthritis, and inflammatory bowel disease. An inactivating E219A mutation, validated by crystallography and NMR spectra, prevents autolysis of MMP-12 and allows us to determine its NMR structure without an inhibitor. The structural ensemble of the catalytic domain without an inhibitor is based on 2813 nuclear Overhauser effects (NOEs) and has an average RMSD to the mean structure of 0.25 Å for the backbone and 0.61 Å for all heavy atoms for residues Trp109-Gly263. Compared to crystal structures of MMP-12, helix B (hB) at the active site is unexpectedly more deeply recessed under the β-sheet. This opens a pocket between hB and β-strand IV in the active-site cleft. Both hB and an internal cavity are shifted toward β-strand I, β-strand III, and helix A on the back side of the protease. About 25 internal NOE contacts distinguish the inhibitor-free solution structure and indicate hB's greater depth and proximity to the sheet and helix A. Line broadening and multiplicity of amide proton NMR peaks from hB are consistent with hB undergoing a slow conformational exchange among subtly different environments. Inhibitor-binding-induced perturbations of the NMR spectra of MMP-1 and MMP-3 map to similar locations across MMP-12 and encompass the internal conformational adjustments. Evolutionary trace analysis suggests a functionally important network of residues that encompasses most of the locations adjusting in conformation, including 18 residues with NOE contacts unique to inhibitor-free MMP-12. The conformational change, sequence analysis, and inhibitor perturbations of NMR spectra agree on the network they identify between structural scaffold and the active site of MMPs.
KW - NMR
KW - conformational adjustment
KW - crystal structure
KW - macrophage elastase
KW - solution structure
UR - http://www.scopus.com/inward/record.url?scp=36248991892&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2007.10.028
DO - 10.1016/j.jmb.2007.10.028
M3 - Article
C2 - 17997411
AN - SCOPUS:36248991892
SN - 0022-2836
VL - 374
SP - 1333
EP - 1344
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -