Biology subjects

Walshe, J. L.

Publications and source records attributed to Walshe, J. L..

7 recordsLinked to original sources

bioRxiv2025

Peptide molecular glues select between BET paralogues by exploiting allosteric sites and conformational dynamics

Achieving selective target inhibition is critical for minimising drug side effects. This can be especially challenging when targeting individual members of protein families with high sequence similarity. A well-recognised example is the Bromodomain and Extraterminal domain (BET) family of proteins. Chemical inhibition of the acetyllysine (AcK)-binding bromodomains (BDs) of BET proteins has shown considerable promise in a range of disease models. However, despite over a decade of medicinal chemistry efforts, it has proven challenging to develop BET BD inhibitors that exhibit high selectivity between BET family paralogues. Cyclic peptides are versatile scaffolds for therapeutic development and often exhibit high selectivity and affinity for their targets. To explore their potential as selective BET BD inhibitors, we have used mRNA display to identify cyclic peptide ligands for the N-terminal BD of BRD2 and BRD4. The structurally diverse cyclic peptides enriched from the selections boast superior selectivity and affinity to previously developed inhibitors. Most strikingly, we isolated cyclic peptides with [~]1000-fold higher affinity for their target BD over the paralogous BDs, far surpassing selectivities reported to date. Our biochemical and structural data suggest that paralogue-selective cyclic peptides act as molecular glues, exploiting both subtle sequence differences at locations far from the AcK-binding pocket and differences in conformational dynamics between BET BD paralogues to achieve this unprecedented level of specificity. This work provides a blueprint for the development of new classes of selective BET inhibitors and, more generally, underscores the potential of exploiting protein dynamics in the design of selective ligands.

biochemistry↗

bioRxiv2024

Molecular mechanism of co-transcriptional H3K36 methylation by SETD2

Tri-methylation of histone H3 at residue lysine-36 (H3K36me3) is a hallmark of actively and recently transcribed genes and contributes to cellular memory and identity. The deposition of H3K36me3 occurs co-transcriptionally when the methyltransferase SETD2 associates with RNA polymerase II (Pol II). Here we present three cryo-EM structures of SETD2 bound to Pol II elongation complexes at different states of nucleosome passage. Together with functional probing, our results suggest a 3-step mechanism of transcription-coupled H3K36me3 deposition. First, binding to the elongation factor SPT6 tethers the catalytic SET domain in proximity to the upstream DNA. Second, Pol II nucleosome passage leads to the transfer of a hexasome from downstream to upstream, poised for methylation. Finally, continued transcription leads to upstream nucleosome reassembly, partial dissociation of the histone chaperone FACT and sequential methylation of both H3 tails, completing H3K36me3 deposition of an upstream nucleosome after Pol II passage.

biochemistry↗

bioRxiv2020

Subunit epsilon of E. coli F1Fo ATP synthase attenuates enzyme activity by modulating central stalk flexibility

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures of E. coli ATP synthase in different rotational and inhibited states observed following a 45 second incubation with 10 mM MgATP. The structures generated describe multiple changes that occur following addition of MgATP, with the inhibitory C-terminal domain of subunit {varepsilon} ({varepsilon}CTD) disassociating from the central stalk to adopt a condensed "down" conformation. The transition to the {varepsilon}CTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by [~]50{degrees}, with further flexing in the peripheral stalk enabling the c-ring to rotate by two sub-steps in the Fo motor. Truncation mutants lacking the second helix of the {varepsilon}CTD suggest that central stalk rotational flexibility is important for F1Fo ATP synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the {varepsilon} subunit.

biophysics↗

bioRxiv2020

Structural characterization of an RNA bound antiterminator protein reveal a successive binding mode

In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in dissociation from the DNA template. ANTAR domains are a class of antiterminator that bind and stabilise mutually exclusive dual hexaloop motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from E. faecialis, both in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate the conformational changes that occur upon RNA binding and reveal the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.

biophysics↗

bioRxiv2019

Cyclic peptides can engage a single binding pocket through multiple, entirely divergent modes

Cyclic peptide display screening techniques can identify drug leads and biological probes with exceptional affinity and specificity. To date, however, the structural and functional diversity encoded in such peptide libraries remains unexplored. We have used the Random nonstandard Peptide Integrated Discovery (RaPID) system to develop cyclic peptide inhibitors of several acetyllysine-binding bromodomains from the Bromodomain and Extra-Terminal domain (BET) family of epigenetic regulators. These peptides have very high affinities for their targets and exhibit extraordinary selectivity (up to 106-fold), making them the highest-affinity and most specific BET-binding molecules discovered to date. Crystal structures of 13 distinct peptide-bromodomain complexes, which all target the acetyllysine-binding pocket, reveal remarkable diversity in both peptide structure and binding mode, and include both -helical and {beta}-sheet type structures. The peptides can exhibit a high degree of structural pre-organization and bivalent binding of two BDs by one peptide was common, flagging the potential for a new direction in inhibitor design that could bring stronger discrimination between BET-family paralogues. Our data demonstrate for the first time the enormous potential held in these libraries to provide a wide array of modes against a single target, maximizing the opportunity to attain high potency and specificity ligands to a wide variety of proteins.

biochemistry↗

bioRxiv2019

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae

Many commensal bacteria and opportunistic pathogens scavenge heme from their environment. Pathogens and host are engaged in an arms race to control access to heme, but similar conflicts between bacterial species that might regulate pathogen colonisation are largely unknown. We show here that a commensal bacterium, Haemophilus haemolyticus, makes hemophilin, a heme-binding protein that not only allows the bacterium to effectively scavenge heme for its own growth, but also inhibits co-culture of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi), by heme starvation. Knockout of the hemophilin gene abrogates the ability of H. haemolyticus to inhibit NTHi and an x-ray crystal structure shows that hemophilin has a previously unreported heme-binding structure. The bound heme molecule is deeply buried and the heme iron atom is coordinated through a single histidine side chain. Biochemical characterization shows that this arrangement allows heme to be captured in the ferrous or ferric state, and with small ferrous or ferric heme-ligands bound, suggesting hemophilin could function over in a wide range of physiological conditions. Our data raise the possibility that competition for heme between commensal and pathogenic bacteria can influence bacterial colonisation, and therefore disease likelihood, and suggest that strains of H. haemolyticus that overproduce hemophilin might have therapeutic uses in reducing colonisation and subsequent opportunistic infection by NTHi.

biochemistry↗

bioRxiv2019

Visualizing movements in E. coli F1Fo ATP synthase indicates how the F1 and Fo motors are coupled

F1Fo ATP synthase functions as a biological rotary generator and makes a major contribution to cellular energy production. It is comprised of two motors that are coupled together by a central \"rotor\" and peripheral \"stator\" stalk. Proton flow through the Fo motor generates rotation of the central stalk that induces conformation changes that catalyze production of ATP in the F1 motor. Here we provide 3-4 [A] resolution cryo-EM structures of E. coli F1Fo ATP synthase in 10 mM MgADP. In addition to generating a comprehensive structural model of E. coli F1Fo ATP synthase to provide a framework to interpret mutagenesis studies, we describe a rotational sub-step of the Fo motor c-ring associated with long-range conformational changes that suggests an elegant mechanism by which the F1 and Fo motors can be coupled with minimal energy loss.

biophysics↗