Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry.
Matrix metalloproteinases (MMPs) play an essential role in normal and pathological extracellular matrix degradation. Deuterium exchange mass spectrometry (DXMS) was used to localize the binding regions of the broad-spectrum MMP inhibitor doxycycline on the active form of matrilysin (residues 95-267) and to assess alterations in structure induced by doxycycline binding. DXMS analyses of inhibitor-bound versus inhibitor-free forms of matrilysin reveal two primary sites of reduced hydrogen/deuterium exchange (residues 145-153; residues 193-204) that flank the structural zinc binding site. Equilibrium dialysis studies of doxycycline-matrilysin binding yielded a K(d) of 73 microM with a binding stoichiometry of 2.3 inhibitor molecules per protein, which compares well with DXMS results that show principal reduction in deuterium exchange at two sites. Lesser changes in deuterium exchange evident at the amino and carboxyl termini are attributed to inhibitor-induced structural fluctuations. Tryptophan fluorescence quenching experiments of matrilysin with potassium iodide suggest changes in conformation induced by doxycycline binding. In the presence of doxycycline, tryptophan quenching is reduced by approximately 17% relative to inhibitor-free matrilysin. Examination of the X-ray crystal structure of matrilysin shows that the doxycycline-binding site at residues 193 to 204 is positioned within the structural metal center of matrilysin, adjacent to the structural zinc atom and near both calcium atoms. These results suggest a mode of matrilysin inhibition by doxycycline that could involve interactions with the structural zinc atom and/or calcium atoms within the structural metal center of the protein.
García
Ricardo A
RA
Department of Medicine, University of California-San Diego, 9500 Gilman Drive, BSB 0613J, La Jolla, CA 92093, USA.
Pantazatos
Dennis P
DP
Gessner
Christopher R
CR
Go
Katrina V
KV
Woods
Virgil L
VL
Villarreal
Francisco J
FJ
D001665:Binding Sites; D041961:Deuterium Exchange Measurement; D004318:Doxycycline / Q000378:metabolism*; D005453:Fluorescence; D013058:Mass Spectrometry; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors / Q000378:metabolism*
D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D020783:Matrix Metalloproteinase 7; D004318:Doxycycline