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Design of activated serine–containing catalytic triads with atomic-level accuracy

Nature Chemical Biology volume 10, pages 386–391 (2014)Cite this article

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Abstract

A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.

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Figure 1: Design strategy.
Figure 2: Design and experimental characterization of OSH55 and related designs.
Figure 3: Organophosphate reactivity of OSH55.4_1 is comparable to that of native enzymes.
Figure 4: Crystal structures of OSH55.4_1 bound to DFP.

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Acknowledgements

We thank K. Masuda and D. Milliken for helping with ABPP screening, R. Xiao and G. Kornhaber for experimental support with sample preparation and structure determination and the Rosen Lab for sharing instrumentation for performing fluopol experiments. This work was supported in part by the Defense Threat Reduction Agency (DTRA) (D.B.), the National Institute on Drug Abuse grant DA033670 (B.F.C.) and by a grant from the National Institute of General Medical Sciences Protein Structure Initiative (PSI), U54-GM094597 (J.F.H.). Fellowship support from the Sir Henry Wellcome Postdoctoral Fellowship (S.R.), NIH–National Institute of Environmental Health Sciences K99/R00 Pathways to Independence Postdoctoral Award 1K99ES020851-01 (C.W.) and Helen Hay Whitney Fellowship (M.L.M.) are gratefully acknowledged.

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Author notes

  1. Florian Richter

    Present address: Present address: Institute of Biology, Humboldt-Universität zu Berlin, Germany.,

  2. Sridharan Rajagopalan and Chu Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, Washington, United States

    Sridharan Rajagopalan, Kai Yu, Florian Richter & David Baker

  2. The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, United States

    Chu Wang, Megan L Matthews & Benjamin F Cravatt

  3. Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York, USA

    Alexandre P Kuzin, Scott Lew, Jayaraman Seetharaman, Min Su & John F Hunt

  4. Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, Washington, USA

    Florian Richter & David Baker

  5. EXCET, Inc., Springfield, Virginia, USA

    Aleksandr E Miklos

  6. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    David Baker

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  1. Sridharan Rajagopalan
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Contributions

S.R., C.W., B.F.C. and D.B. conceived the project. S.R. and F.R. performed the computational design, S.R. expressed and purified the designed proteins. S.R. and K.Y. performed the yeast display experiments. C.W. performed the ABPP screening and mass spec experiments. C.W. and M.L.M. performed the fluopol experiments. A.P.K., A.E.M., S.L., J.S., M.S. and J.F.H. performed the crystallography experiments. S.R., C.W., B.F.C. and D.B. analyzed data and wrote the manuscript.

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Correspondence to David Baker.

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The authors declare no competing financial interests.

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Rajagopalan, S., Wang, C., Yu, K. et al. Design of activated serine–containing catalytic triads with atomic-level accuracy. Nat Chem Biol 10, 386–391 (2014). https://doi.org/10.1038/nchembio.1498

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