Biology subjects

Walkinshaw, M. D.

Publications and source records attributed to Walkinshaw, M. D..

4 recordsLinked to original sources

bioRxiv2024

Allosteric inhibition of trypanosomatid pyruvate kinases by a camelid single-domain antibody

African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

biochemistry↗

bioRxiv2018

Atomic Level Characterisation of Millisecond-Time Scale Protein Motions through a Combined Molecular Simulations and NMR Approach

Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they inter-convert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov State modelling (MSM) to explore these excited conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1{rho} solution state NMR measurements. By accurately and reliably exploring functionally relevant, but sparsely populated conformations with milli-second lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery.

biophysics↗

bioRxiv2017

Pushing The Limits Of Detection Of Weak Binding Using Fragment Based Drug Discovery: Identification Of New Cyclophilin Binders

Fragment Based Drug Discovery (FBDD) is an increasingly popular method to identify novel small-molecule drug candidates. One of the limitations of the approach is the difficulty of accurately characterizing weak binding events. This work reports a combination of X-ray diffraction, surface plasmon resonance (SPR) experiments and molecular dynamics (MD) simulations, for the characterisation of binders to different isoforms of the cyclophilin (Cyp) protein family. Although several Cyp inhibitors have been reported in the literature, it has proven challenging to achieve high binding selectivity for different isoforms of this protein family. The present studies have led to the identification of several structurally novel fragments that bind to diverse Cyp isoforms in distinct pockets with low millimolar dissociation constants. A detailed comparison of the merits and drawbacks of the experimental and computational techniques is presented, and emerging strategies for designing ligands with enhanced isoform specificity are described.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC=\"FIGDIR/small/136101_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@5d2d48org.highwire.dtl.DTLVardef@147f922org.highwire.dtl.DTLVardef@356562org.highwire.dtl.DTLVardef@1933853_HPS_FORMAT_FIGEXP M_FIG C_FIG Research Highlights O_LIFBDD is a popular method but weak binding is difficult to detect\nC_LIO_LIThere is a need for pushing the limits of weak binding detection\nC_LIO_LICombination of X-ray, SPR and MD methodologies increases successful characterization of weak binding events\nC_LIO_LISeveral novel Cyclophilin fragment binders were identified\nC_LI

biophysics↗

bioRxiv2016

Domains of methylated CAC and CG target MeCP2 to tune transcription in the brain

Mutations in the gene encoding the methyl-CG binding protein MeCP2 cause neurological disorders including Rett syndrome. The di-nucleotide methyl-CG (mCG) is the canonical MeCP2 DNA recognition sequence, but additional targets including non-methylated sequences have been reported. Here we use brain-specific depletion of DNA methyltransferase to show that DNA methylation is the primary determinant of MeCP2 binding in mouse brain. In vitro and in vivo analyses reveal that MeCP2 binding to non-CG methylated sites in brain is largely confined to the tri-nucleotide sequence mCAC. Structural modeling suggests that mCG and mCAC may be interchangeable as minimal structural perturbation of MeCP2 accompanies binding. MeCP2 binding to chromosomal DNA in mouse brain is proportional to mCG + mCAC density and defines domains within which transcription is sensitive to MeCP2 occupancy. The results suggest that MeCP2 interprets patterns of mCAC and mCG in the brain to negatively modulate transcription of genes critical for neuronal function.

molecular biology↗