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Lookup NU author(s): Dr Owen Burbidge, Dr Martyna PastokORCiD, Dr Diana PapiniORCiD, Kenneth Tan, Dr Shannon TurbervilleORCiD, Professor Martin NobleORCiD, Professor Jane EndicottORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2025 The Author(s)We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 is a cell cycle regulator that inhibits cyclin-dependent kinases CDK4 and CDK6 and is inactivated in sporadic and familial cancers. The majority of p16 missense mutations cause loss of function by destabilizing the protein's structure. We identify nanobodies that bind p16 with nanomolar affinities and restore the stability of many different cancer-associated p16 mutations located at sites throughout the protein. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. We confirm that nanobodies bind to p16 in a cellular setting and do not preclude p16 binding to CDK6 and its ability to induce cell-cycle arrest. These findings indicate that nanobodies merit testing as pharmacological chaperones for p16 reactivation in the cell.
Author(s): Burbidge O, Pastok MW, Papini D, Hodder SL, Zenkeviciute G, Tan KZJ, Turberville S, Noble MEM, Endicott JA, Itzhaki L
Publication type: Article
Publication status: Published
Journal: Structure
Year: 2025
Pages: epub ahead of print
Online publication date: 14/08/2025
Acceptance date: 20/07/2025
Date deposited: 01/09/2025
ISSN (print): 0969-2126
ISSN (electronic): 1878-4186
Publisher: Cell Press
URL: https://doi.org/10.1016/j.str.2025.07.017
DOI: 10.1016/j.str.2025.07.017
Data Access Statement: All crystallographic results have been deposited at the Protein DataBank (PDB) https://www.rcsb.org/and are publicly available. Accession numbers are listed in the key resources table. PDB accession codes for structures used within, but not derived from, this study, CDK6-p16INK4a (PDB: 1BI715), GFP:GFP nanobody complex (PDB:3OGO58), p16INK4A (PDB 2A5E24). Biophysical assays (nanobody analysis by circular dichroism, urea and thermal denaturation) and HTRF, SPR, ITC, SEC, DSF and thermal profiling data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
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