Biology subjects

Toso, D.

Publications and source records attributed to Toso, D..

5 recordsLinked to original sources

bioRxiv2021

Structural basis for the ARF GAP activity and specificity of the C9orf72 complex

Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required [~]10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP.

biochemistry↗

bioRxiv2021

Efficient graphene oxide coating improves cryo-EM sample preparation and data collection from tilted grids

Recent technical developments have made single particle cryo-EM a major structural biology technique, especially in the characterization of challenging samples that resist crystallization, can only be obtained in small amounts, or suffer from compositional or conformational heterogeneity. However, a number of hurdles that often challenge sample preparation still need to be overcome in order to increase the applicability and throughput of cryo-EM. These technical hurdles include obtaining enough particles per image, with close to random orientation, and without damage from interaction with the air-water interface. While coating EM grids with graphene oxide is a promising procedure for the improvement of sample preparation, it suffers from some technical problems that limit its applicability. We have modified the established drop cast method for adhering graphene oxide onto holey patterned grids to increase graphene coverage. Our method relies on the use of a polycationic polymer to coat the surface of the grid prior to graphene oxide application, thereby preventing the repulsion of the negatively charged graphene oxide sheets from the negatively charged grid surface. With this improved preparation method, we show that graphene oxide supports can increase the number of particles in the field of view by an order of magnitude with respect to open holes, while keeping them away from the damaging air-water interface. We also show how graphene oxide coated gold foil grids can be used to collect tilted cryo-EM data in order to overcome preferred orientation issues, without experiencing the large amount of drift observed with conventional amorphous carbon supports, thus allowing data collection that can lead to high-resolution reconstructions.

biophysics↗

bioRxiv2020

JARID2 and AEBP2 regulate PRC2 activity in the presence of H2A ubiquitination or other histone modifications

The Polycomb repressive complexes PRC1 and PRC2 functionally interact to coordinate cell type identity by the epigenetic regulation of gene expression. It has been proposed that PRC2 is recruited to genomic loci via the recognition of PRC1-mediated mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub1), but the mechanism of this process remains poorly understood. Here, we report the cryo-EM structure of human PRC2 with cofactors JARID2 and AEBP2 bound to a nucleosome substrate containing H2AK119ub1. We find that JARID2 and AEBP2 each interact with one of the two ubiquitin molecules in the nucleosome. A ubiquitin-interaction motif (UIM) in JARID2 is sandwiched between ubiquitin and the histone H2A-H2B acidic patch. Simultaneously, the tandem zinc-fingers of AEBP2 interact with the second ubiquitin and the histone H2A-H2B surface on the opposite side of the nucleosome. JARID2 plays a dual role in the H2AK119ub1 dependent stimulation of PRC2 through interactions with both EED via its K116 trimethylation and with the H2AK119-ubiquitin. AEBP2, on the other hand, appears to primarily serve as a scaffold contributing to the interaction between PRC2 and the H2AK119ub1 nucleosome. Our structure also provides a detailed visualization of the EZH2-nucleosome interface, revealing a segment of EZH2 (named "bridge helix") that is stabilized as it bridges the EZH2(SET) domain, the H3 tail and the nucleosomal DNA. In addition to the role played by AEBP2 and JARID2 in PRC2 regulation by H2AK119ub1 recognition, we also observe that the presence of these cofactors partially overcomes the inhibitory effect that H3K4- and H3K36-trimethylation have on core PRC2. Together, our results reveal the central role played by cofactors JARID2 and AEBP2 in orchestrating the crosstalk between histone post-translational modifications and PRC2 methyltransferase activity.

biophysics↗

bioRxiv2019

Effects of α-tubulin acetylation on microtubule structure and stability

Acetylation of K40 in -tubulin is the sole post-translational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy reconstructions of pure samples of TAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as the structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains K40. Modification of K40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.

biophysics↗

bioRxiv2018

The complete structure of the human TFIIH core complex

Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the architecture of TFIIH and its interactions within transcription pre-initiation complexes. Here, we present the complete structure of the human TFIIH core complex, determined by phase-plate cryo-electron microscopy at 3.7 [A] resolution. The structure uncovers the molecular basis of TFIIH assembly, revealing how the recruitment of XPB by p52 depends on a pseudo-symmetric dimer of homologous domains in these two proteins. The structure also suggests a function for p62 in the regulation of XPD, and allows the mapping of previously unresolved human disease mutations.

biophysics↗