Biology subjects

Bridges, H. R.

Publications and source records attributed to Bridges, H. R..

5 recordsLinked to original sources

bioRxiv2026

Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures

Mycobacteria synthesise the unusual glycan [x1D05]-arabinan as a major component of the cell wall glycoconjugates arabinogalactan (AG) and lipoarabinomannan (LAM). We previously identified Dysgonomonas gadei, a member of the Bacteroidota, as capable of complete [x1D05]-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo--[x1D05]-arabinofuranosidase activity against AG and LAM, although their linkage specificities were unknown. Here, using defined synthetic substrates, we show that the three enzymes possess distinct linkage preferences. We also develop -[x1D05]-arabinofuranosyl cyclophellitol aziridines as covalent inhibitors and activity-based probes for GH172 enzymes. X-ray crystallography and cryo-EM to reveal strikingly different quaternary assemblies across the three homologues, while a 1.5 [A] cryo-EM structure of dodecameric Dg67 covalently modified by an aziridine inhibitor identifies the catalytic nucleophile and provides direct structural support for a retaining mechanism. A BODIPY-tagged aziridine probe selectively labelled the three GH172 enzymes in D. gadei cell lysates. Together, these findings define functional and structural diversity within GH172 and establish chemical probes for profiling -[x1D05]-arabinofuranosidase activity in complex biological samples.

biochemistry↗

bioRxiv2025

ALC1 Finds a New Foothold on the Nucleosome's Super-Groove

Nucleosomes act as recognition platforms for chromatin-binding factors that coordinate genome maintenance. A distinct structural feature of the nucleosome is the alignment of the DNA major and minor grooves across both gyres, forming continuous major and minor super-grooves. Despite this prominent feature, only one example of a naturally occurring protein binding to a super-groove is documented at the structural level to date. The chromatin remodeler Amplified in Liver Cancer 1 (ALC1) is a key component of the DNA damage response and a promising therapeutic target in cancer. Through extensive classification of our cryo-electron microscopy data, we identified a previously unresolved ALC1-nucleosome complex characterized by a loosely bound conformation of ALC1. This conformation represents an intermediate between the auto-inhibited and active states, and provides new structural insights into the conformational transitions that regulate ALC1 activity. In this intermediate state, the linker of ALC1 engages the nucleosomes minor DNA super-groove across both gyres in a recognition mode observed for the first time in a naturally occurring protein. SynopsisRe-analysis of a publicly available cryo-EM dataset identifies an intermediate state of the oncogenic chromatin remodeler ALC1, revealing the conformational change leading to its activation upon binding to a PARylated nucleosome. The remodeler recognizes the nucleosomes super-groove.

biophysics↗

bioRxiv2024

Structure of the turnover-ready state of an ancestral respiratory complex I

Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-[A] resolution structure of complex I from the -proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.

biochemistry↗

bioRxiv2022

Structural Basis of Mammalian Respiratory Complex I Inhibition by Medicinal Biguanides

The molecular mode of action of metformin, a biguanide used widely in the treatment of diabetes, is incompletely characterized. Here we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo-electron microscopy and enzyme kinetics. We explain the unique selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel and an additional binding site is in a pocket on the intermembrane space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable rational design of novel medicinal biguanides. One-Sentence SummaryBiguanides inhibit complex I by binding in the quinone channel, and exert an independent localized chaotropic effect.

biochemistry↗

bioRxiv2022

Cryo-EM structures reveal ubiquinone-10 binding to mitochondrial complex I and conformational transitions associated with Q-site occupancy

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, structures of Q10-bound states have remained elusive. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the active (ready-to-go) resting state (plus a matching substrate-free structure) and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the deactive resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.

biochemistry↗