Biology subjects

Manfield, I. W.

Publications and source records attributed to Manfield, I. W..

4 recordsLinked to original sources

bioRxiv2026

Selective miniprotein inhibitors of Aurora-A kinase designed using interaction-motif scaffolding

Targeting kinase ATP-binding sites has produced many successful therapeutics, but selectivity remains a major challenge. We hypothesised that substrate-recognition surfaces could provide an alternative route to selective kinase inhibition. Here, we designed and tested structure-guided miniprotein inhibitors of Aurora-A using N-Myc as a template, a natural weak binder of the P+1 pocket. The most potent designs bind Aurora-A with single-digit nanomolar affinity, more than a thousand fold higher than the starting template, and selectively inhibit Aurora-A over Aurora-B in kinase assays and mitotic cells. Despite diverse architectures, successful binders targeting the substrate-recognition surface converged on a common strategy, coupling extensive engagement of the G helix with activation loop stabilisation. Together these results show that selective kinase inhibition can be achieved by exploiting structurally well-defined active-state conformations rather than kinase-specific inactive states. Our work establishes a framework for converting weak substrate-recognition surface interactors into potent, selective kinase inhibitors through structure-guided protein design.

molecular biology↗

bioRxiv2024

Structurally diverse calloses/β-1,3-glucans in plant cell wall microdomains

Cell walls underpin the mechanics of cell growth, intercellular signalling, and defence against pathogenic organisms. {beta}-(1,3)-glucans (also known as callose) are polysaccharides found in plants, fungi, and some bacterial species. In developing plant organs, callose accumulates around intercellular channels (plasmodesmata) controlling cell-to-cell communication. We developed monoclonal antibodies for the detection of {beta}-(1,3)-glucans and using these identified distinct populations of callose differing in size and secondary structure. Callose sub-populations were in proximal but not overlapping cell wall microdomains implying distinct spatial and functional microenvironments. We also unveiled callose interaction with xyloglucan; another plant glycan regulating cell wall functions. This work challenges previous views demonstrating structural heterogeneity in plant callose and supporting interactions between glycans with roles in the regulation of cell wall properties and functions.

plant biology↗

bioRxiv2024

Molecular chaperone BiP controls activity of the ER stress sensor Ire1 through interactions with its oligomers

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

biophysics↗

bioRxiv2019

A fuzzy encounter complex precedes formation of the fully-engaged TIR1-Aux/IAA auxin co-receptor system

The plant hormone auxin regulates almost every aspect of plant development via the TIR1/AFB-auxin-Aux/IAA auxin co-receptor complex. Within this ternary complex, auxin acts as a molecular glue to promote the binding of Aux/IAA transcriptional repressor proteins to SCFTIR1/AFB ubiquitin-ligase complexes, thereby catalysing their ubiquitin-mediated proteolysis. A conspicuous feature of the crystal structure of the complex is a rare cis W-P bond within the Aux/IAA degron motif. To study receptor complex assembly, we have used NMR to determine the solution structure of the amino-terminal half of the Aux/IAA protein AXR3/IAA17, including the degron, both in isolation and in complex with TIR1 and auxin. We show that this region of AXR3 is intrinsically-disordered with only limited elements of structure and yet the critical degron W-P bond occurs with an unusually high (1:1) ratio of cis to trans isomers. We show that assembly of the co-receptor complex involves both auxin-dependent and -independent interaction events in which the disorder of the Aux/IAA is retained. Further, using the synthetic auxin molecule cvxIAA and by analysing specific Aux/IAA conformers, we show that a subset of auxin-dependent binding events occur away from the base of the canonical auxin binding pocket in TIR1. Our results reveal the existence of a fuzzy, topologically-distinct ternary encounter complex and thus that auxin perception is not limited to sequential, independent binding of auxin and then Aux/IAA to TIR1.

plant biology↗