Biology subjects

Beton, J. G.

Publications and source records attributed to Beton, J. G..

5 recordsLinked to original sources

bioRxiv2024

Stepwise recruitment of Hsc70 by DNAJB1 produces ordered arrays primed for bursts of amyloid fibre disassembly

To understand the action of co-chaperones of the J-domain protein family in the assembly of disaggregation-active Hsc70 complexes on the surface of amyloid fibres, we used cryo-EM and tomography to compare the assemblies with wild-type co-chaperones or an activity deficient mutant. We show that human DNAJB1 binds uniformly and densely along -synuclein amyloid fibres in an asymmetric orientation, with one subunit of the DNAJB1 dimer lying along the fibre surface and the other subunit further away. It acts in a 2-step recruitment of Hsc70 to the fibres, first releasing DNAJB1 auto-inhibition and then activating the Hsc70 ATPase by the J domain, with ATPase recycling stimulated by the nucleotide exchange factor Apg2. When the auto-inhibition is removed by mutating the H5 inhibitory binding site on DNAJB1 ({Delta}1H5 DNAJB1 mutant), Hsc70 is recruited to the fibres at a normal level, but the resulting complex is inactive in disaggregation. Cryo tomography of the wild-type DNAJB1:Hsc70:Apg2:Syn fibre complex shows dense arrays of the chaperones extending out from the fibre surface along spiral tracks, with the Hsc70 density on the outer surface of the DNAJB1 layer. The {Delta}1H5 DNAJB1:Hsc70:Apg2:Syn fibre complex results in equally dense but less organised binding all over the fibre surface and lacks the ordered clusters. On the basis of these findings, we propose that 2-step activation of DNAJB1 regulates Hsc70 access to the fibre substrate. Since the DNAJB1 dimers are bound every 40 [A], the J domain released by Hsc70 binding to one dimer could activate the ATPase of an Hsc70 bound to the adjacent dimer. This could trigger a cascade of recruitment and activation in a localised, dense cloud of Hsc70 molecules to give coordinated, sequential binding and disaggregation from an exposed fibre end, as observed by cryo-EM and earlier fluorescence and AFM studies.

biochemistry↗

bioRxiv2023

CASP15 cryoEM protein and RNA targets: refinement and analysis using experimental maps

CASP assessments primarily rely on comparing predicted coordinates with experimental reference structures. However, errors in the reference structures can potentially reduce the accuracy of the assessment. This issue is particularly prominent in cryoEM-determined structures, and therefore, in the assessment of CASP15 cryoEM targets, we directly utilized density maps to evaluate the predictions. A method for ranking the quality of protein chain predictions based on rigid fitting to experimental density was found to correlate well with the CASP assessment scores. Overall, the evaluation against the density map indicated that the models are of high accuracy although local assessment of predicted side chains in a 1.52 [A] resolution map showed that side-chains are sometimes poorly positioned. The top 136 predictions associated with 9 protein target reference structures were selected for refinement, in addition to the top 40 predictions for 11 RNA targets. To this end, we have developed an automated hierarchical refinement pipeline in cryoEM maps. For both proteins and RNA, the refinement of CASP15 predictions resulted in structures that are close to the reference target structure, including some regions with better fit to the density. This refinement was successful despite large conformational changes and secondary structure element movements often being required, suggesting that predictions from CASP-assessed methods could serve as a good starting point for building atomic models in cryoEM maps for both proteins and RNA. Loop modeling continued to pose a challenge for predictors with even short loops failing to be accurately modeled or refined at times. The lack of consensus amongst models suggests that modeling holds the potential for identifying more flexible regions within the structure.

bioinformatics↗

bioRxiv2021

Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of -synuclein, huntingtin or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Recruitment and clustering of Hsc70 on the fibrils appear to be necessary for disassembly. Here we use atomic force microscopy (AFM) to show that segments of in vitro assembled -synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography reveals fibrils with regions of densely bound chaperones extending from the fibre surface, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of [~]4 subunits per second.

biochemistry↗

bioRxiv2020

TopoStats - an automated tracing program for AFM images

We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, 90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub (https://github.com/afm-spm/TopoStats) and is intended to be modified and adapted for use if required.

biophysics↗

bioRxiv2019

Combining high-resolution AFM with MD simulations shows that DNA supercoiling induces kinks and defects that enhance flexibility and recognition.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

biophysics↗