A global survey of intramolecular isopeptide bonds

Costa, Francesco, Riziotis, Ioannis, Andreeva, Antonina, Kalwan, Delhi, de Jong, Jennifer, Hinchliffe, Philip, Parmeggiani, Fabio ORCID: https://orcid.org/0000-0001-8548-1090, Race, Paul R., Burston, Steven G., Bateman, Alex and Barringer, Rob 2025. A global survey of intramolecular isopeptide bonds. Protein Science 34 (12) , e70342. 10.1002/pro.70342

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Abstract

Many proteins harbor covalent intramolecular bonds that enhance their stability and resistance to thermal, mechanical, and proteolytic insults. Intramolecular isopeptide bonds represent one such covalent interaction, yet their distribution across protein domains and organisms has been largely unexplored. Here, we sought to address this by employing a large‐scale prediction of intramolecular isopeptide bonds in the AlphaFold database using the structural template‐based software Isopeptor. Our findings reveal an extensive phyletic distribution in bacterial and archaeal surface proteins resembling fibrillar adhesins and pilins. All identified intramolecular isopeptide bonds are found in two structurally distinct folds, CnaA‐like or CnaB‐like, from a relatively small set of related Pfam families, including 10 novel families that we predict to contain intramolecular isopeptide bonds. One CnaA‐like domain of unknown function, DUF11 (renamed here to “CLIPPER”) is broadly distributed in cell‐surface proteins from Gram‐positive bacteria, Gram‐negative bacteria, and archaea, and is structurally and biophysically characterized in this work. Using x‐ray crystallography, we resolve a CLIPPER domain from a Gram‐negative fibrillar adhesin that contains an intramolecular isopeptide bond and further demonstrate that it imparts thermostability and resistance to proteolysis. Our findings demonstrate the extensive distribution of intramolecular isopeptide bond‐containing protein domains in nature and structurally resolve the previously cryptic CLIPPER domain.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Schools > Pharmacy
Additional Information:	License information from Publisher: LICENSE 1: URL: http://creativecommons.org/licenses/by/4.0/
Publisher:	Wiley
ISSN:	0961-8368
Date of First Compliant Deposit:	18 November 2025
Date of Acceptance:	2 October 2025
Last Modified:	18 Nov 2025 16:15
URI:	https://orca.cardiff.ac.uk/id/eprint/182493

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