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Emergence of specific binding and catalysis from a designed generalist binding protein

Nature Chemistry (2026) Cite this article

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Abstract

Binding and catalysis play central roles in living systems. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for the incremental evolution of different specificities. Thus, modern proteins have emerged from the joint exploration of sequence and structural space. Interactions between natural proteins and small molecules can be systematically profiled by crystallographic fragment screening in defined geometries, yet this approach has not been applied to highly designable de novo proteins. Here we apply this method to explore the binding specificity of a de novo small-molecule-binding protein, apixaban-binding helical bundle. As in nature, we found that it formed weak complexes, which were excellent starting points for the design of entirely distinct functions, including a turn-on fluorophore binder and a highly efficient Kemp eliminase with a catalytic efficiency of 3,200,000 M−1 s−1, approaching the diffusion limit. This work illustrates how simultaneous consideration of sequence and chemical structure diversity can guide the emergence of different functions in designed proteins.

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Fig. 1: Nature-inspired evolution of de novo proteins.
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Fig. 2: Design of FABLE binding to fluorogenic coumarins.
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Fig. 3: Design of an efficient KABLE and its directed evolution.
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Fig. 4: NMR-guided directed evolution of KABLE1.4.
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Fig. 5: The origin of KABLE’s efficient catalysis probed by X-ray crystallography and MD simulation.
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Fig. 6: Exploration of sequence and chemical space of de novo protein–ligand interactions for the design of distinctive functions.
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Data availability

The crystal structures of ABLE–fragment complexes have been deposited in the PDB with accession codes 7HIY, 7HIZ, 7HJ0, 7HJ1, 7HJ2, 7HJ3, 7HJ4, 7HJ5, 7HJ6, 7HJ7, 7HJ8, 7HJ9, 7HJA, 7HJB, 7HJC, 7HJD, 7HJE, 7HJF, 7HJG, 7HJH, 7HJI, 7HJJ, 7HJK, 7HJL, 7HJM, 7HJN, 7HJO, 7HJP, 7HJQ, 7HJR, 7HJS, 7HJT, 7HJU, 7HJV, 7HJW, 7HJX, 7HJY, 7HJZ, 7HK0, 7HK1, 7HK2, 7HK3 and 7HK4. A summary of data collection, modelling and refinement of ABLE–fragment complexes is provided in Supplementary Information. Structure factor intensities (unmerged, merged and merged/scaled), PanDDA input and output files (including Z-map and event maps in CCP4 format), and refined models (including the fragment-bound state extracted from multistate models) are available via Zenodo at https://doi.org/10.5281/zenodo.13913848 (ref. 116). The crystal structures of FABLE and KABLE have been deposited in the PDB with accession codes 9DWA, 9DWB, 9DWC, 9N0I and 9N0J. X-ray data collection and refinement statistics for the FABLE and KABLE structures are provided in Supplementary Information. All other data not included in the article and Supplementary Information are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

Customized Python scripts used for protein design and modelling are provided in Supplementary Information. These Python scripts are also publicly available via Zenodo at https://doi.org/10.5281/zenodo.17935960 (ref. 117).

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Acknowledgements

We thank a reviewer for suggesting the direct comparison of HG3.17 with KABLE2.5. We are grateful for helpful discussions with S. Schneider and H. Jo. We thank members of the DeGrado and Fraser labs for support. The synchrotron X-ray diffraction data used to determine the crystal structures reported in this Article were collected at beamline 8.3.1 of the Advanced Light Source (ALS) and beamlines 12-1 and 12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL). We acknowledge the use of the Wynton high-performance computer cluster at UCSF. We are grateful to the National Science Foundation (CHE-2108660 and MCB- 2306190, to W.F.D.) and the National Institutes of Health (R35GM122603, to W.F.D.) for funding. J.S.F. was supported by a Sanghvi-Agarwal Innovation Award and the National Institutes of Health (NIH GM145238). W.F.D. thanks the W. M. Keck Foundation for its support. Use of the SSRL, SLAC National Accelerator Laboratory, was supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-76SF00515). The SSRL Structural Molecular Biology Program is supported by the US DOE, Office of Biological and Environmental Research and the National Institutes of Health, National Institute of General Medical Sciences (grant no. P30GM133894). The ALS, a US DOE Office of Science User Facility (contract no. DE-AC02-05CH11231), is supported in part by the ALS-ENABLE program funded by the NIH, National Institute of General Medical Sciences (grant no. P30GM124169). N.F.P. acknowledges funding from the NIH (R00GM135519). J.E.G. acknowledges funding from the NIH (R01GM141299). I.V.K. is thankful for funding support from the National Institute of Health (NIH R35GM119634) and Welch Foundation (AA-2198-20240404). S.B. is the Connie and Bob Lurie Fellow of the Damon Runyon Cancer Research Foundation (DRG-2522-24). S.B. is a recipient of a postdoctoral independent research grant (7032759) from the Program for Breakthrough Biomedical Research, which the Sandler Foundation partially funds. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Author notes

  1. These authors contributed equally: Yuda Chen, Sagar Bhattacharya, Lena Bergmann, Galen J. Correy.

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

    Yuda Chen, Sagar Bhattacharya, Sophia K. Tan, Kaipeng Hou, Lei Lu, Ian Bakanas, Jason E. Gestwicki & William F. DeGrado

  2. Cardiovascular Research Institute, University of California, San Francisco, CA, USA

    Yuda Chen, Sagar Bhattacharya, Sophia K. Tan, Kaipeng Hou, Lei Lu, Ian Bakanas & William F. DeGrado

  3. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA

    Lena Bergmann, Galen J. Correy, Justin T. Biel & James S. Fraser

  4. Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, USA

    Jason E. Gestwicki

  5. VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium

    Alexander N. Volkov

  6. Jean Jeener NMR Centre, Vrije Universiteit Brussel (VUB), Brussels, Belgium

    Alexander N. Volkov

  7. Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA

    Ivan V. Korendovych

  8. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Nicholas F. Polizzi

  9. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Nicholas F. Polizzi

  10. Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan

    William F. DeGrado

Authors
  1. Yuda Chen
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  2. Sagar Bhattacharya
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Contributions

Y.C., S.B., N.F.P., J.S.F. and W.F.D. formulated the project. L.B., G.J.C. and J.T.B. performed the fragment screening. Y.C. performed the computational design and carried out the experimental characterization of FABLEs. Y.C. conducted the computational design of KABLEs. S.B. and Y.C. carried out the experimental optimization and characterization of KABLE. S.B. conducted the directed evolution of KABLE. A.N.V. collected the NMR spectra. L.B., G.J.C. and Y.C. obtained the crystal structure data for the FABLEs and KABLEs. Y.C. and S.K.T. performed the MD simulations. K.H., L.L. and I.B. assisted with the computational design. Y.C., S.B., G.J.C., K.H., J.E.G., I.V.K., N.F.P., J.S.F. and W.F.D. wrote the paper with input from all authors.

Corresponding authors

Correspondence to James S. Fraser or William F. DeGrado.

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Competing interests

J.S.F. has an equity in and is a compensated consultant for Profluent Bio. The other authors declare no competing interests.

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Nature Chemistry thanks Shuguang Zhang 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 X-ray crystal structure of ABLE showing binding sites for 43 fragments.

The structure of apo ABLE (PDB: 9DW2) with white cartoon and transparent white surface.

Extended Data Fig. 2 Chemical structures and electron density maps for the ABLE-fragment complex structures (PDBs: 7HJA, 7HJ2, 7HJL, 7HJY, 7HK3, 7HK1, 7HK2, 7HJH, 7HIY, 7HIZ, 7HJV, 7HJC.

PanDDA event maps (blue mesh, 2 σ) are contoured around fragments (teal/yellow sticks). The sidechains of residues Tyr46 and His49 are shown with purple sticks. Fragment names, PDB codes, resolution, 1-BDC value and refined occupancies are indicated. Hydrogen bonds are shown with dashed black lines. For clarity, residues 10–23 and 105–118 are hidden for fragments binding in the ABLE core. His49-mediated polar interaction could be found in the majority of fragments at site B.

Extended Data Fig. 3 Chemical structures and electron density maps for the ABLE-fragment complex structures (PDBs: 7HJI, 7HJ0, 7HJQ, 7HJ4, 7HJ5, 7HJZ, 7HK0,7HK4, 7HJG, 7HJJ, 7HJX, 7HJF).

PanDDA event maps (blue mesh, 2 σ) are contoured around fragments (teal/yellow sticks). The sidechains of residues Tyr46 and His49 are shown with purple sticks. Fragment names, PDB codes, resolution, 1-BDC value and refined occupancies are indicated. Hydrogen bonds are shown with dashed black lines. For clarity, residues 10–23 and 105–118 are hidden for fragments binding in the ABLE core. His49-mediated polar interaction could be found in the majority of fragments at site B.

Extended Data Fig. 4 Chemical structures and electron density maps for the ABLE-fragment complex structures (PDBs: 7HJK, 7HJE, 7HJ9, 7HJW, 7HJB, 7HJ7, 7HJ8, 7HJ3, 7HJ6, 7HJD, 7HJM, 7HJS).

PanDDA event maps (blue mesh, 2 σ) are contoured around fragments (teal/yellow sticks). The sidechains of residues Tyr46 and His49 are shown with purple sticks. Fragment names, PDB codes, resolution, 1-BDC value and refined occupancies are indicated. Hydrogen bonds are shown with dashed black lines. For clarity, residues 10–23 and 105–118 are hidden for fragments binding in the ABLE core. His49-mediated polar interaction could be found in the majority of fragments at site B.

Extended Data Fig. 5 Chemical structures and electron density maps for the ABLE-fragment complex structures (PDBs: 7HJP,7HJU,7HJR, 7HJ1,7HJO,7HJN,7HJT).

PanDDA event maps (blue mesh, 2 σ) are contoured around fragments (teal/yellow sticks). The sidechains of residues Tyr46 and His49 are shown with purple sticks. Fragment names, PDB codes, resolution, 1-BDC value and refined occupancies are indicated. Hydrogen bonds are shown with dashed black lines. For clarity, residues 10–23 and 105–118 are hidden for fragments binding in the ABLE core. His49-mediated polar interaction could be found in the majority of fragments at site B.

Extended Data Fig. 6 Contour map of Tyr46 side chain conformation of ABLE from molecular dynamics.

(a-b) Dynamics of uncomplexed ABLE (PDB: 6W6X) and ABLE-apixaban (PDB: 6W70) were explored by doing molecular simulation at 278 K for 500 ns with Amber. Chi1 and Chi2 from each state of these simulations and reported crystal structures of ABLEs (6W6X: uncompleted ABLE; 6W70: ABLE-Apixaban Complex; 6X8N: uncomplexed ABLE His49Ala) were extracted for plotting. Contours were generated from the frames of uncomplexed ABLE (a) and ABLE-apixaban complex contours (b) using an in-house script that utilizes the Gaussian kernel density estimate methods from scipy.stats module of SciPy Python Packages. (c-d) A and B conformations of Tyr46 sidechain were present in the uncomplexed ABLE crystal structure (c, PDB: 6W6X), while only A conformation of Tyr46 sidechain conformation was present at ABLE-apixaban complex (d, PDB: 6W70).

Extended Data Fig. 7 Characterization of five FABLE Designs.

Left column: Fluorescence of Cou485 at two fixed concentrations of 3 μM and 6 μM were measured in the presence of increasing amounts of each protein. The dissociation constant was obtained by globally fitting a single-site binding model to data, as described at Supplementary Methods. The error bars represent standard deviations of three independent measurements. Middle Column: circular dichroism spectra shows that all the designs are helical proteins. Right column: temperature-dependent circular dichroism signals measured at 222 nm show that all designs are thermostable. The design 1 was designated as FABLE.

Extended Data Fig. 8 Exploring the chemical space of FABLE.

The excitation and emission of fluorophores are in Supplementary Table 3. The error bars represent standard deviations of three measurements.

Extended Data Table 1 Kinetic parameters for KABLEs
Full size table
Extended Data Table 2 Kinetic parameters of reported proteins catalyzing the Kemp elimination of 5-nitrobenzisoxazole with the base-mediated mechanism
Full size table

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–23, Tables 1–12, Methods, Notes and References.

Reporting Summary (download PDF )

Supplementary Table 1 (download XLSX )

X-ray data collection and analysis.

Source data

Source Data Fig. 2 (download XLSX )

Numerical data for the plots in Fig. 2.

Source Data Fig. 3 (download XLSX )

Numerical data for the plots in Fig. 3.

Source Data Fig. 4 (download XLSX )

Numerical data for the plots in Fig. 4.

Source Data Fig. 5 (download XLSX )

Numerical data for the plots in Fig. 5.

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Chen, Y., Bhattacharya, S., Bergmann, L. et al. Emergence of specific binding and catalysis from a designed generalist binding protein. Nat. Chem. (2026). https://doi.org/10.1038/s41557-026-02125-6

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