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
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
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
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).
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).
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).
Miller, W. T.Determinants of substrate recognition in nonreceptor tyrosine kinases. Acc. Chem. Res. 36, 393–400 (2003).
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).
Scott, M. P. & Miller, W. T. A peptide model system for processive phosphorylation by Src family kinases. Biochemistry 39, 14531–14537 (2000).
Qiu, H. & Miller, W. T. Role of the Brk SH3 domain in substrate recognition. Oncogene 23, 2216–2223 (2004).
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).
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).
Park, S.-H., Zarrinpar, A. & Lim, W. A. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299, 1061–1064 (2003).
Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).
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).
Bolukbasi, M. F. et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015).
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).
Dyla, M. & Kjaergaard, M. Intrinsically disordered linkers control tethered kinases via effective concentration. Proc. Natl Acad. Sci. USA 117, 21413–21419 (2020).
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).
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).
Grove, T. L. et al. Structural insights into thioether bond formation in the biosynthesis of sactipeptides. J. Am. Chem. Soc. 139, 11734–11744 (2017).
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).
Cao, L. et al. De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426–431 (2020).
Alford, R. F. et al. The Rosetta all-atom energy function for macromolecular modeling and design. J. Chem. Theory Comput. 13, 3031–3048 (2017).
Karanicolas, J. et al. A de novo protein binding pair by computational design and directed evolution. Mol. Cell 42, 250–260 (2011).
Maguire, J. B. et al. Perturbing the energy landscape for improved packing during computational protein design. Proteins 89, 436–449 (2021).
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).
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).
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).
Plaxco, K. W. et al. The folding kinetics and thermodynamics of the Fyn–SH3 domain. Biochemistry 37, 2529–2537 (1998).
Johnson, K. A. New standards for collecting and fitting steady state kinetic data. Beilstein J. Org. Chem. 15, 16–29 (2019).
Goldsmith, M. & Tawfik, D. S. Enzyme engineering: reaching the maximal catalytic efficiency peak. Curr. Opin. Struct. Biol. 47, 140–150 (2017).
Tianero, M. D. et al. Metabolic model for diversity-generating biosynthesis. Proc. Natl Acad. Sci. USA 113, 1772–1777 (2016).
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).
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).
Cho, K. F. et al. Split-TurboID enables contact-dependent proximity labeling in cells. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020).
Rivera, V. M. et al. A humanized system for pharmacolog ic control of gene expression. Nat. Med. 2, 1028–1032 (1996).
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).
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).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).
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).
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
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
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.
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.
About this article
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 19, 460–467 (2023). https://doi.org/10.1038/s41589-022-01206-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41589-022-01206-0
This article is cited by
-
Peptides hit the catalysis walk
Nature Chemical Biology (2023)