Biology subjects

Davey, N. E.

Publications and source records attributed to Davey, N. E..

25 records · Page 2Linked to original sources

bioRxiv2021

A structural biology community assessment of AlphaFold 2 applications

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods have led to protein structure predictions that have reached the accuracy of experimentally determined models. While this has been independently verified, the implementation of these methods across structural biology applications remains to be tested. Here, we evaluate the use of AlphaFold 2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modelling of interactions; and modelling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modelled when compared to homology modelling, identifying structural features rarely seen in the PDB. AF2-based predictions of protein disorder and protein complexes surpass state-of-the-art tools and AF2 models can be used across diverse applications equally well compared to experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life science research.

biophysics↗

bioRxiv2021

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, current methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a scalable viral peptide discovery approach covering 229 RNA viruses that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an {Phi}xFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its {Phi}xFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction blocks SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

biochemistry↗

bioRxiv2021

Proteome-scale amino-acid resolution footprinting of protein-binding sites in the intrinsically disordered regions of the human proteome

Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies using a wide range of experimental approaches have identified tens of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type-specific interactions are likely to be disproportionately under-represented. Moreover, for most known protein-protein interactions the binding regions remain uncharacterized. We previously developed proteomic peptide phage display (ProP-PD), a method for simultaneous proteome-scale identification of short linear motif (SLiM)-mediated interactions and footprinting of the binding region with amino acid resolution. Here, we describe the second-generation human disorderome (HD2), an optimized ProP-PD library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay. We define guidelines for how to process, filter and rank the results and provide PepTools, a toolkit for annotation and analysis of identified hits. We uncovered 2,161 interaction pairs for 35 known SLiM-binding domains and confirmed a subset of 38 interactions by biophysical or cell-based assays. Finally, we show how the amino acid resolution binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the human proteome. The HD2 ProP-PD library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.

biochemistry↗

bioRxiv2020

Comprehensive analysis of fungal G1 cyclin docking motif sequences that control CDK regulatory potency in vivo

Cyclin-dependent kinases (CDKs) control the ordered series of events during eukaryotic cell division. The stage at which individual CDK substrates are phosphorylated can be dictated by cyclin-specific docking motifs. In budding yeast, substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs and the conservation of this mechanism were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with mutational scanning and deep sequencing. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results reveal the basis for variations in potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the potency of other candidate motifs. In one protein, Whi5, we found overlapping LP and phosphorylation motifs with partly redundant effects. In another protein, the CDK inhibitor Sic1, we found that its LP motif is inherently weak due to unfavorable residues at key positions, and this imposes a beneficial delay in its phosphorylation and degradation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in LP motif potency can tune the strength and timing of CDK regulation.

molecular biology↗

bioRxiv2019

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins - structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

cell biology↗

bioRxiv2019

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

PP1 and PP2A-B56 are major serine/threonine phosphatase families that achieve specificity by colocalising with substrates. At the kinetochore, however, both phosphatases localise to an almost identical molecular space and yet they still manage to regulate unique pathways and processes. By switching or modulating the positions of PP1/PP2A-B56 at kinetochores, we show that their unique downstream effects are not due to either the identity of the phosphatase or its precise location. Instead, these phosphatases signal differently because their kinetochore recruitment can be either inhibited (PP1) or enhanced (PP2A) by phosphorylation inputs. Mathematical modelling explains how these inverse phospho-dependencies elicit unique forms of cross-regulation and feedback, which allows otherwise indistinguishable phosphatases to produce distinct network behaviours and control different mitotic processes. Therefore, the kinetochore uses PP1 and PP2A-B56 because their binding motifs respond to kinase inputs in opposite ways. Genome-wide motif analysis suggests that many other pathways also select for these same key features, implying that these similar catalytic enzymes may have diverged during evolution to allow opposite modes of phospho-regulation.

cell biology↗

bioRxiv2018

Conserved SQ and QS motifs in bacterial effectors suggest pathogen interplay with the ATM kinase family during infection

Understanding how bacteria hijack eukaryotic cells during infection is vital to develop better strategies to counter the pathologies that they cause. ATM kinase family members phosphorylate eukaryotic protein substrates on Ser or Thr residues followed by Gln. The kinases are active under oxidative stress conditions and/or the presence of ds-DNA breaks. While examining the protein sequences of well-known bacterial effector proteins such as CagA and Tir, we noticed that they often show conserved (S/TQ) motifs, even though the evidence for effector phosphorylation by ATM has not been reported. We undertook a bioinformatics analysis to examine effectors for their potential to mimic the eukaryotic substrates of the ATM kinase. The candidates we found could interfere with the hosts intracellular signaling network upon interaction, which might give an advantage to the pathogen inside the host. Further, the putative phosphorylation sites should be accessible, conserved across species and, in the vicinity to the phosphorylation sites, positively charged residues should be depleted. We also noticed that the reverse motif (QT/S) is often also conserved and located close to (S/TQ) sites, indicating its potential biological role in ATM kinase function. Our findings could suggest a mechanism of infection whereby many pathogens inactivate/modulate the host ATM signaling pathway.

bioinformatics↗