Biology subjects

Burston, S. G.

Publications and source records attributed to Burston, S. G..

3 recordsLinked to original sources

bioRxiv2025

A global survey of intramolecular isopeptide bonds

Many protein domains harbour 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 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 ten 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 characterised 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.

bioinformatics↗

bioRxiv2024

Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4+2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

biochemistry↗

bioRxiv2022

High resolution analysis of proteolytic substrate processing

Proteolysis is a key catalytic event in protein and thus cellular homeostasis. Despite the importance and wide implications of proteolytic processing and degradation, methods describing the degradation of folded proteins at high temporal and spatial resolution are not well established. However, this information is required to obtain a deep mechanistic understanding of proteolytic events and their consequences. Here, we describe an integrated method comprising time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics to reveal the sequential degradation and unfolding of the model substrate annexin A1 by the human serine protease HTRA1. This workflow represents a general strategy for obtaining precise molecular insights into protease-substrate interactions that can be conveniently adapted to studying other posttranslational modifications such as phosphorylation in dynamic protein complexes.

biochemistry↗