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Title: Receptor subtype discrimination using extensive shape complementary designed interfaces

Abstract

To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. Here, we describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.

Authors:
 [1]; ORCiD logo [2];  [3];  [3];  [4];  [5]; ORCiD logo [6];  [6];  [3];  [3];  [3];  [7];  [3];  [6]; ORCiD logo [1]
  1. Univ. of Washington, Seattle, WA (United States). Dept. of Biochemistry; Univ. of Washington, Seattle, WA (United States). Inst. for Protein Design; Univ. of Washington, Seattle, WA (United States). Howard Hughes Medical Inst.
  2. Stanford Univ., CA (United States). Dept. of Molecular and Cellular Physiology; Stanford Univ., CA (United States). School of Medicine, Dept. of Structural Biology; Stanford Univ., CA (United States). Howard Hughes Medical Inst.
  3. Stanford Univ., CA (United States). Dept. of Medicine, Division of Hematology
  4. Korea Inst. of Science and Technology, Gangneung (China). Systems Biotechnology Research Center
  5. Stanford Univ., CA (United States). Dept. of Molecular and Cellular Physiology; Stanford Univ., CA (United States). School of Medicine, Dept. of Structural Biology; Stanford Univ., CA (United States). Howard Hughes Medical Inst.; Princess Máxima Center for Pediatric Oncology, Utrecht (The Netherlands)
  6. Stanford Univ., CA (United States). Dept. of Molecular and Cellular Physiology; Stanford Univ., CA (United States). School of Medicine, Dept. of Structural Biology; Stanford Univ., CA (United States). Howard Hughes Medical Inst.
  7. Stanford Univ., CA (United States). School of Medicine, Dept. of Comparative Medicine
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)
OSTI Identifier:
1532481
Grant/Contract Number:  
AC02-76SF00515; AC02-06CH11357; AC02-05CH11231; 1S10OD012289-01A1; U01DK085527; U19AI116484; R01NS100904; U01CA217851
Resource Type:
Accepted Manuscript
Journal Name:
Nature Structural & Molecular Biology
Additional Journal Information:
Journal Volume: 26; Journal Issue: 6; Journal ID: ISSN 1545-9993
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 97 MATHEMATICS AND COMPUTING

Citation Formats

Dang, Luke T., Miao, Yi, Ha, Andrew, Yuki, Kanako, Park, Keunwan, Janda, Claudia Y., Jude, Kevin M., Mohan, Kritika, Ha, Nhi, Vallon, Mario, Yuan, Jenny, Vilches-Moure, José G., Kuo, Calvin J., Garcia, K. Christopher, and Baker, David. Receptor subtype discrimination using extensive shape complementary designed interfaces. United States: N. p., 2019. Web. doi:10.1038/s41594-019-0224-z.
Dang, Luke T., Miao, Yi, Ha, Andrew, Yuki, Kanako, Park, Keunwan, Janda, Claudia Y., Jude, Kevin M., Mohan, Kritika, Ha, Nhi, Vallon, Mario, Yuan, Jenny, Vilches-Moure, José G., Kuo, Calvin J., Garcia, K. Christopher, & Baker, David. Receptor subtype discrimination using extensive shape complementary designed interfaces. United States. doi:10.1038/s41594-019-0224-z.
Dang, Luke T., Miao, Yi, Ha, Andrew, Yuki, Kanako, Park, Keunwan, Janda, Claudia Y., Jude, Kevin M., Mohan, Kritika, Ha, Nhi, Vallon, Mario, Yuan, Jenny, Vilches-Moure, José G., Kuo, Calvin J., Garcia, K. Christopher, and Baker, David. Mon . "Receptor subtype discrimination using extensive shape complementary designed interfaces". United States. doi:10.1038/s41594-019-0224-z. https://www.osti.gov/servlets/purl/1532481.
@article{osti_1532481,
title = {Receptor subtype discrimination using extensive shape complementary designed interfaces},
author = {Dang, Luke T. and Miao, Yi and Ha, Andrew and Yuki, Kanako and Park, Keunwan and Janda, Claudia Y. and Jude, Kevin M. and Mohan, Kritika and Ha, Nhi and Vallon, Mario and Yuan, Jenny and Vilches-Moure, José G. and Kuo, Calvin J. and Garcia, K. Christopher and Baker, David},
abstractNote = {To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. Here, we describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.},
doi = {10.1038/s41594-019-0224-z},
journal = {Nature Structural & Molecular Biology},
number = 6,
volume = 26,
place = {United States},
year = {2019},
month = {5}
}

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    Works referencing / citing this record:

    Wnt/β-Catenin Signaling in Development and Disease
    journal, November 2006


    Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities
    journal, June 2017


    Apc Tumor Suppressor Gene Is the “Zonation-Keeper” of Mouse Liver
    journal, June 2006


    The IFN-λ-IFN-λR1-IL-10Rβ Complex Reveals Structural Features Underlying Type III IFN Functional Plasticity
    journal, March 2017


    From DARPins to LoopDARPins: Novel LoopDARPin Design Allows the Selection of Low Picomolar Binders in a Single Round of Ribosome Display
    journal, February 2014

    • Schilling, Johannes; Schöppe, Jendrik; Plückthun, Andreas
    • Journal of Molecular Biology, Vol. 426, Issue 3
    • DOI: 10.1016/j.jmb.2013.10.026

    Wnt signalling in stem cells and cancer
    journal, April 2005


    Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche
    journal, March 2009

    • Sato, Toshiro; Vries, Robert G.; Snippert, Hugo J.
    • Nature, Vol. 459, Issue 7244
    • DOI: 10.1038/nature07935

    Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling
    journal, May 2017

    • Janda, Claudia Y.; Dang, Luke T.; You, Changjiang
    • Nature, Vol. 545, Issue 7653
    • DOI: 10.1038/nature22306

    Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal
    journal, May 2017

    • Yan, Kelley S.; Janda, Claudia Y.; Chang, Junlei
    • Nature, Vol. 545, Issue 7653
    • DOI: 10.1038/nature22313

    Comprehensive analysis of kinase inhibitor selectivity
    journal, October 2011

    • Davis, Mindy I.; Hunt, Jeremy P.; Herrgard, Sanna
    • Nature Biotechnology, Vol. 29, Issue 11
    • DOI: 10.1038/nbt.1990

    Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing
    journal, May 2012

    • Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan
    • Nature Biotechnology, Vol. 30, Issue 6
    • DOI: 10.1038/nbt.2214

    Yeast surface display for screening combinatorial polypeptide libraries
    journal, June 1997

    • Boder, Eric T.; Wittrup, K. Dane
    • Nature Biotechnology, Vol. 15, Issue 6
    • DOI: 10.1038/nbt0697-553

    High-affinity binders selected from designed ankyrin repeat protein libraries
    journal, April 2004

    • Binz, H. Kaspar; Amstutz, Patrick; Kohl, Andreas
    • Nature Biotechnology, Vol. 22, Issue 5, p. 575-582
    • DOI: 10.1038/nbt962

    Wnt signalling induces maturation of Paneth cells in intestinal crypts
    journal, March 2005

    • van Es, Johan H.; Jay, Philippe; Gregorieff, Alex
    • Nature Cell Biology, Vol. 7, Issue 4
    • DOI: 10.1038/ncb1240

    Computational design of self-assembling cyclic protein homo-oligomers
    journal, December 2016

    • Fallas, Jorge A.; Ueda, George; Sheffler, William
    • Nature Chemistry, Vol. 9, Issue 4
    • DOI: 10.1038/nchem.2673

    A liver Hif-2α–Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition
    journal, September 2013

    • Wei, Kevin; Piecewicz, Stephanie M.; McGinnis, Lisa M.
    • Nature Medicine, Vol. 19, Issue 10
    • DOI: 10.1038/nm.3295

    Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors
    journal, July 2012

    • Gurney, A.; Axelrod, F.; Bond, C. J.
    • Proceedings of the National Academy of Sciences, Vol. 109, Issue 29
    • DOI: 10.1073/pnas.1120068109

    Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries
    journal, July 2012

    • Kummer, L.; Parizek, P.; Rube, P.
    • Proceedings of the National Academy of Sciences, Vol. 109, Issue 34
    • DOI: 10.1073/pnas.1205399109

    Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1
    journal, December 2003

    • Kuhnert, F.; Davis, C. R.; Wang, H. -T.
    • Proceedings of the National Academy of Sciences, Vol. 101, Issue 1
    • DOI: 10.1073/pnas.2536800100

    Regulation of retinal progenitor expansion by Frizzled receptors: implications for microphthalmia and retinal coloboma
    journal, January 2012

    • Liu, Chunqiao; Bakeri, Hirva; Li, Tiansen
    • Human Molecular Genetics, Vol. 21, Issue 8
    • DOI: 10.1093/hmg/ddr616

    Integration, scaling, space-group assignment and post-refinement
    journal, January 2010

    • Kabsch, Wolfgang
    • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 133-144
    • DOI: 10.1107/s0907444909047374

    Features and development of Coot
    journal, March 2010

    • Emsley, P.; Lohkamp, B.; Scott, W. G.
    • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
    • DOI: 10.1107/s0907444910007493

    Towards automated crystallographic structure refinement with phenix.refine
    journal, March 2012

    • Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
    • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
    • DOI: 10.1107/s0907444912001308

    Structural Basis of Wnt Recognition by Frizzled
    journal, May 2012


    Structural basis for Notch1 engagement of Delta-like 4
    journal, February 2015


    Notch Signaling: Cell Fate Control and Signal Integration in Development
    journal, April 1999


    Insights into Cytokine–Receptor Interactions from Cytokine Engineering
    journal, March 2015


    Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy
    journal, January 2015


    Tgf-β Signal Transduction
    journal, June 1998


    Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts
    journal, May 2014

    • Kabiri, Z.; Greicius, G.; Madan, B.
    • Development, Vol. 141, Issue 11
    • DOI: 10.1242/dev.104976