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Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

Nature Structural & Molecular Biology volume 18, pages 1102–1108 (2011)Cite this article

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Abstract

Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

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Figure 1: Schematic illustration of the experimental setup to measure structural kinetics and solvation dynamics of metalloenzymes in real time.
Figure 2: Real-time spectroscopic analysis of metalloenzyme (MT1-MMP) catalysis of the peptide substrate Mca-PLGL(Dnp)AR.
Figure 3: Analysis of time-dependent X-ray absorption spectra.
Figure 4: Changes in hydration dynamics are mostly associated with Michaelis complex formation.
Figure 5: Gradient of coupled protein-water motions.

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Acknowledgements

We thank A. Frenkel (Yeshiva University), J. Bohon, M. Sullivan (beam line X3B at the National Synchrotron Light Source), I. Solomonov and Y. Udi (Weizmann Institute of Science) for help with X-ray absorption data collection, and we thank M. Krüger (Ruhr-University Bochum) for programming the THz data acquisition software. We thank G. Murphy (Cambridge Research Institute) for the plasmid encoding TIMP-2. We acknowledge financial support by the Ministry of Innovation, Science, Research and Technology of the German state of North Rhine-Westphalia and by the Ruhr-University Bochum and thank the Ressourcenverbund North Rhine-Westphalia for computer time. B.B. and M.He. were members of the Ruhr-University Research School funded by Germany's Excellence Initiative (DFG GSC 98/1). B.B. is grateful to the Feinberg Graduate School at the Weizmann Institute for a Dean of Faculty fellowship. M.He. was a fellow of the Studienstiftung des Deutschen Volkes. G.B.F. is supported by the Robert A. Welch Foundation. G.B.F. and I.S. are supported by a US National Institutes of Health grant (CA098799). I.S. is supported by the Israel Science Foundation, the Kimmelman Center at the Weizmann Institute and the Ambach family fund. M.Ha. is supported by the VW Stiftung.

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Author notes

  1. Moran Grossman, Benjamin Born, Matthias Heyden, Irit Sagi and Martina Havenith: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Structural Biology, The Weizmann Institute of Science, Rehovot, Israel

    Moran Grossman, Dmitry Tworowski & Irit Sagi

  2. Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel

    Moran Grossman & Irit Sagi

  3. Lehrstuhl für Physikalische Chemie II, Ruhr-Universität Bochum, Bochum, Germany

    Benjamin Born, Matthias Heyden & Martina Havenith

  4. Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas, USA

    Gregg B Fields

  5. The Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida, USA

    Gregg B Fields

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Contributions

I.S. and M.Ha. are equal contributors and designed the experiments, analyzed the data and wrote the manuscript. M.G., B.B. and M.He. are equal contributors and conducted the research, analyzed the data and wrote the manuscript. D.T. constructed the enzyme-substrate docking model. G.B.F. provided the substrates.

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Correspondence to Irit Sagi or Martina Havenith.

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Grossman, M., Born, B., Heyden, M. et al. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site. Nat Struct Mol Biol 18, 1102–1108 (2011). https://doi.org/10.1038/nsmb.2120

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