Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 Scaffold
Open accessAbstract
The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. We diversified positions on a β-sheet and surface loops that together form a concave surface. The new library produced binding proteins (termed "monobodies") to multiple target proteins, generally with similar efficacy as the original, loop-focused library. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics.
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POI-targeting polypeptides can either be single chain antibodies (e.g. nanobodies) or a variety of synthetic polypeptide scaffolds that bind POIs such as monobodies, DARPINs, affimers, or Affibodies.
“These POI-targeting polypeptides can either be single chain antibodies (e.g. variable domains from camelid or shark-derived heavy chain only antibodies, nanobodies), or a variety of synthetic polypeptide scaffolds that bind POIs such as fibronectin type III domain-based monobodies [110, 111], designed ankyrin repeats (DARPINs) [112, 113], cystatin-derived affimers [114] or Affibodies derived from”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [111]
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- [27]Gilbreth RN Esaki K Koide A Sidhu SS Koide S 2008 A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces J Mol Biol 381 407 18 18602117 PMC2582520
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- Authors
- Koide, A., Wojcik, J., Gilbreth, R. N., Hoey, R. J., & Koide, S.
- Affiliations
- University of Chicago (US)
- Corresponding author
- Koide, Shohei
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2012-01
- Volume
- 415
- Issue
- 2
- Pages
- 393-405
- ISSN
- 0022-2836 (ISSN-L)
- PMC
- PMC3260337
- OpenAlex
- W2092499431
- Cited by
- 227
- References
- 54
- Citations by year
- 2026: 13 · 2025: 8 · 2024: 21 · 2023: 27 · 2022: 15 · 2021: 14 · 2020: 20 · 2019: 8 · 2018: 23 · 2017: 19 · 2016: 14 · 2015: 18 · 2014: 11 · 2013: 12 · 2012: 4
- FWCI
- 6.61
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (NIH) (R01-GM072688, R01-GM090324, 5T32GM07281-33, 085P1000817, DE-AC02-06CH11357)
- University of Chicago Comprehensive Cancer Center
- NIH
- National Center for Research Resources
- Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor
- U.S. Department of Energy, Office of Basic Energy Sciences
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- monobody, fibronectin type III domain, combinatorial protein libraries, concave surfaces, protein-protein interaction
- MeSH terms
- Amino Acid Sequence, Fibronectins, Models, Molecular, Molecular Sequence Data, Protein Binding, Sequence Homology, Protein Structure, Tertiary, Crystallography, X-Ray, Peptide Library, Protein Structure, Quaternary, Mutant Proteins
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