Molecular structures and dynamics of the stepwise activation mechanism of a matrix metalloproteinase zymogen: challenging the cysteine switch dogma.
Activation of matrix metalloproteinase zymogen (pro-MMP) is a vital homeostatic process, yet its molecular basis remains unresolved. Using stopped-flow X-ray spectroscopy of the active site zinc ion, we determined the temporal sequence of pro-MMP-9 activation catalyzed by tissue kallikrein protease in milliseconds to several minutes. The identity of three intermediates seen by X-ray spectroscopy was corroborated by molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. The cysteine-zinc interaction that maintains enzyme latency is disrupted via active-site proton transfers that mediate transient metal-protein coordination events and eventual binding of water. Unexpectedly, these events ensue as a direct result of complexation of pro-MMP-9 and kallikrein and occur before proteolysis and eventual dissociation of the pro-peptide from the catalytic site. Here we demonstrate the synergism among long-range protein conformational transitions, local structural rearrangements, and fine atomic events in the process of zymogen activation.
Rosenblum
Gabriel
G
Department of Structural Biology, The Weizmann Institute of Science, Rehovot, Israel.
Meroueh
Samy
S
Toth
Marta
M
Fisher
Jed F
JF
Fridman
Rafael
R
Mobashery
Shahriar
S
Sagi
Irit
I
D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry*; D020782:Matrix Metalloproteinases / Q000737:chemistry*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis / Q000379:methods; D013816:Thermodynamics; D020840:Tissue Kallikreins / Q000737:chemistry; D014965:X-Rays; D015032:Zinc / Q000737:chemistry
D004792:Enzyme Precursors; D020840:Tissue Kallikreins; D020782:Matrix Metalloproteinases; D015032:Zinc; D003545:Cysteine