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Designer installation of a substrate recruitment domain to tailor enzyme specificity

Nature Chemical Biology volume 19, pages 460–467 (2023)Cite this article

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Abstract

Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catechol O-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.

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Fig. 1: General scaffolding diagrams and Rosetta design approach.
Fig. 2: Structural characterization and background activity of the designed enzymes.
Fig. 3: Steady-state kinetics analysis of the designed enzymes.
Fig. 4: Substrate competition experiment.
Fig. 5: Numerical modeling of competing substrates reacting with the engineered enzymes.

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Data availability

Atomic coordinates for the designed SH3–COMT fusion, SH3-588, have been deposited to the Protein Data Bank under the accession number 7UD6. The plasmid for SH3-588 has been deposited to AddGene under plasmid number 185920 (pMCSG28-SH3-588). Source data are provided as Supplementary Data 1.

Code availability

Code used to model kinetic and substrate competition data is available in the Supplementary Information. Rosetta scripts used to design the protein interfaces are also provided in the Supplementary Information and a demo folder containing scripts and input files can also be found in the Supplementary Information.

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Acknowledgements

We thank members of the laboratories of A.A.B. and S.L. Campbell for sharing advice and equipment. In addition, we thank D. Thieker, J. Maguire and A. Leaver-Fay for invaluable advice in creating the design protocol for the multidomain enzyme. This work was supported by NIH grant R35GM131923 (to B.K.) and is based on work supported in part by a discovery grant from the Eshelman Institute for Innovation and National Science Foundation under grant number 2204094 (to A.A.B.).

Author information

Author notes

  1. Chayanid Ongpipattanakul

    Present address: School of Pharmacy, University of California San Francisco, San Francisco, CA, USA

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Rodney Park & Brian Kuhlman

  2. Department of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Chayanid Ongpipattanakul & Satish K. Nair

  3. Center for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Satish K. Nair

  4. Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Satish K. Nair

  5. Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Albert A. Bowers

  6. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Brian Kuhlman

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Contributions

R.P. designed SH3-588 and conducted all of the experimental enzymatic assays. C.O. and S.K.N. crystalized and determined the structure of the designed protein and contributed to the Methods. R.P. and B.K. wrote and tested the custom MATLAB scripts for substrate occlusion. R.P., A.A.B. and B.K. wrote the manuscript and contributed intellectually to the design of the system.

Corresponding authors

Correspondence to Albert A. Bowers or Brian Kuhlman.

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Nature Chemical Biology thanks Andrew Buller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expanded set of timepoints for substrate competition assay.

Expanded set of timepoints for substrate competition assay for target peptide (red) and off-target (light blue) at 1 (a), 10 (b), and 100 (c) μM peptide substrate (2, 20, and 200 μM total peptide respectively). Raw data points are represented by black dots superimposed on plots. Data bars present mean values +/− SEM across three distinct reactions (n = 3, frequently hidden under data points). Error bars are centered on the mean. (d) Exact timepoints collected for 1, 10, and 100 μM. Timepoint 2 is displayed in Fig. 4 in main text and t1 at 100 μM is used in competition reaction mathematical modeling.

Extended Data Fig. 2 Full computational model diagram.

Full computational model diagram. (a) Single, directed substrate simulation, where substrate can bind to the active site and peptide binding site. (b) Multi-substrate (directed and undirected competition) reaction simulation diagram.

Extended Data Fig. 3 Substrate diversity assay.

Substrate diversity assay. (a) Diagram of assay procedure. Briefly, kit components were combined with genes encoding peptides. After IVT incubation, the crude mixture was split, respective enzyme added, and reactions were run at 30 °C for 15 minutes. Samples were prepped and run for MALDI-TOF-MS analysis. (b) Table of IVT peptide substrates tested and their corresponding conversions. SH3-588 largely reached completion for most peptides tested (marked with 100%); remaining substrate peaks were hardly above noise (IVT peptides 1–9). IVT peptides 1–10 were analyzed by MALDI-TOF-MS. Error values indicate +/− SEM across three distinct replicates (n = 3) for IVT peptides 1–8 and two distinct replicates (n = 2) for IVT peptides 9 and 10 centered on the mean.

Supplementary information

Supplementary Information

Supplementary Tables 1–7 and Figs. 1–6, computational protocols and scripts.

Reporting Summary

Supplementary Data 1

Rosetta scripts for the design and docking protocols and MATLAB scripts for the numerical integration models.

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Park, R., Ongpipattanakul, C., Nair, S.K. et al. Designer installation of a substrate recruitment domain to tailor enzyme specificity. Nat Chem Biol 19, 460–467 (2023). https://doi.org/10.1038/s41589-022-01206-0

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