Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Designer installation of a substrate recruitment domain to tailor enzyme specificity

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.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

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.

References

  1. Ho, S. H. & Tirrell, D. A. Enzymatic labeling of bacterial proteins for super-resolution imaging in live cells. ACS Cent. Sci. 5, 1911–1919 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2, 99–104 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Bhattacharyya, R. P., Reményi, A., Yeh, B. J. & Lim, W. A.Domains, motifs, and scaffolds: The role of modular interactions in the evolution and wiring of cell signaling circuits. Annu. Rev. Biochem. 75, 655–680 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Miller, W. T.Determinants of substrate recognition in nonreceptor tyrosine kinases. Acc. Chem. Res. 36, 393–400 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pellicena, P., Stowell, K. R. & Miller, W. T. Enhanced phosphorylation of Src family kinase substrates containing SH2 domain binding sites. J. Biol. Chem. 273, 15325–15328 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Scott, M. P. & Miller, W. T. A peptide model system for processive phosphorylation by Src family kinases. Biochemistry 39, 14531–14537 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Qiu, H. & Miller, W. T. Role of the Brk SH3 domain in substrate recognition. Oncogene 23, 2216–2223 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Ortega, M. A. & van der Donk, W. A. New insights into the biosynthetic logic of ribosomally synthesized and post-translationally modified peptide natural products. Cell Chem. Biol. 23, 31–44 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Park, S.-H., Zarrinpar, A. & Lim, W. A. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299, 1061–1064 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Bolukbasi, M. F. et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bashor, C. J., Helman, N. C., Yan, S. & Lim, W. A. Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics. Science 319, 1539–1543 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Dyla, M. & Kjaergaard, M. Intrinsically disordered linkers control tethered kinases via effective concentration. Proc. Natl Acad. Sci. USA 117, 21413–21419 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Speltz, E. B. & Zalatan, J. G. The relationship between effective molarity and affinity governs rate enhancements in tethered kinase–substrate reactions. Biochemistry 59, 2182–2193 (2020).

    Article  CAS  PubMed  Google Scholar 

  17. Burkhart, B. J., Hudson, G. A., Dunbar, K. L. & Mitchell, D. A.A prevalent peptide-binding domain guides ribosomal natural product biosynthesis. Nat. Chem. Biol. 11, 564–570 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Grove, T. L. et al. Structural insights into thioether bond formation in the biosynthesis of sactipeptides. J. Am. Chem. Soc. 139, 11734–11744 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Leaver-Fay, A. et al. Chapter nineteen—ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cao, L. et al. De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426–431 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Alford, R. F. et al. The Rosetta all-atom energy function for macromolecular modeling and design. J. Chem. Theory Comput. 13, 3031–3048 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Karanicolas, J. et al. A de novo protein binding pair by computational design and directed evolution. Mol. Cell 42, 250–260 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Maguire, J. B. et al. Perturbing the energy landscape for improved packing during computational protein design. Proteins 89, 436–449 (2021).

    Article  CAS  PubMed  Google Scholar 

  24. Camara-Artigas, A., Ortiz-Salmeron, E., Andujar-Sánchez, M., Bacarizo, J. & Martin-Garcia, J. M. The role of water molecules in the binding of class I and II peptides to the SH3 domain of the Fyn tyrosine kinase. Acta Crystallogr. F Struct. Biol. Commun. 72, 707–712 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lotta, T. et al. Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme. Biochemistry 34, 4202–4210 (1995).

    Article  CAS  PubMed  Google Scholar 

  26. Struck, A.-W. et al. An enzyme cascade for selective modification of tyrosine residues in structurally diverse peptides and proteins. J. Am. Chem. Soc. 138, 3038–3045 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Plaxco, K. W. et al. The folding kinetics and thermodynamics of the Fyn–SH3 domain. Biochemistry 37, 2529–2537 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Johnson, K. A. New standards for collecting and fitting steady state kinetic data. Beilstein J. Org. Chem. 15, 16–29 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Goldsmith, M. & Tawfik, D. S. Enzyme engineering: reaching the maximal catalytic efficiency peak. Curr. Opin. Struct. Biol. 47, 140–150 (2017).

    Article  CAS  PubMed  Google Scholar 

  30. Tianero, M. D. et al. Metabolic model for diversity-generating biosynthesis. Proc. Natl Acad. Sci. USA 113, 1772–1777 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Krishnamurthy, V. M., Semetey, V., Bracher, P. J., Shen, N. & Whitesides, G. M. Dependence of effective molarity on linker length for an intramolecular protein–ligand system. J. Am. Chem. Soc. 129, 1312–1320 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Meneses, E. & Mittermaier, A. Electrostatic interactions in the binding pathway of a transient protein complex studied by NMR and isothermal titration calorimetry. J. Biol. Chem. 289, 27911–27923 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cho, K. F. et al. Split-TurboID enables contact-dependent proximity labeling in cells. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Rivera, V. M. et al. A humanized system for pharmacolog ic control of gene expression. Nat. Med. 2, 1028–1032 (1996).

    Article  CAS  PubMed  Google Scholar 

  35. Yazawa, M., Sadaghiani, A. M., Hsueh, B. & Dolmetsch, R. E. Induction of protein–protein interactions in live cells using light. Nat. Biotechnol. 27, 941–945 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Lerner, C. et al. Design of potent and druglike nonphenolic inhibitors for catechol O-methyltransferase derived from a fragment screening approach targeting the S-adenosyl-l-methionine pocket. J. Med. Chem. 59, 10163–10175 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  PubMed  Google Scholar 

  43. Hussain, M., Cummins, M. C., Endo-Streeter, S., Sondek, J. & Kuhlman, B. Designer proteins that competitively inhibit Gαq by targeting its effector site. J. Biol. Chem. 297, 101348 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

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

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Park, R., Ongpipattanakul, C., Nair, S.K. et al. Designer installation of a substrate recruitment domain to tailor enzyme specificity. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01206-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41589-022-01206-0

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing