Biology subjects

Papai, G.

Publications and source records attributed to Papai, G..

4 recordsLinked to original sources

bioRxiv2025

Affinity-ligand purification of native human low-abundance multi-protein complexes for structure determination

Human SAGA is a 20-subunit complex that stimulates transcription and is essential for development. The most prominent addition to SAGA in metazoans compared to yeast is a 150kDa splicing-factor module (SPL). SPL is also a part of the U2snRNP but its role in SAGA is elusive, partially due to absence of high-resolution structural information regarding its incorporation into the complex. In yeast, subunit TAF5 and TAF6 of SAGA are shared with the general transcription factor TFIID. In metazoans, gene duplication created proteins that occur only in SAGA (TAF5L and TAF6L) or in TFIID (TAF5 and TAF6). What functions of SAGA benefit from this protein specialization is unclear. Here we report the structure of endogenous human SAGA purified via an affinity-ligand from cells that were not disturbed by any genomic engineering tools such as CRISPR-Cas9. Our work reveals the high-resolution structure of SPL and of the TAF6L HEAT repeat domain that provides the SPL with a docking surface. We elucidate how SPL and the HEAT repeats are incorporated into SAGA. We find multiple major differences between TAF6L/TAF5L and the canonical paralogues that are directly implicated in structural re-arrangements required to accommodate SPL. Furthermore, SPL binding to SAGA is very different and occupying much less interaction surface than to U2snRNP. However, the two cases still share similar sequences in a helix that is deeply inserted into the SPL. The seemingly weaker interaction of SPL with SAGA raises the possibility that SAGA serves to relay this module to the splicing machinery. Our structure also suggests mutations that could uncouple SPL from SAGA to further interrogate the role of this module.

biochemistry↗

bioRxiv2025

Specific binding modes of SIRT6 C-terminal domain to the nucleosome core particle influence DNA unwrapping and H3K27 accessibility

Sirtuins are a class of NAD-dependent histone deacetylases that regulate important biological pathways in prokaryotes and eukaryotes. This enzyme family comprises seven members, named SIRT1 to SIRT7. Among them, Sirtuin 6 (SIRT6) is a human sirtuin that deacetylates histones and plays a key role in DNA repair, telomere maintenance, carbohydrate and lipid metabolism, and lifespan. SIRT6s structure consists of a zinc finger domain, a Rossmann fold domain containing the NAD+ binding site, and disordered N-terminal and C-terminal (CTD) extensions. The specific role of the CTD on SIRT6 interaction with nucleosomes for histone deacetylation remains unclear. Here, we resort to extended molecular dynamics simulations to uncover the dynamical behavior of the full-length SIRT6 bound to a nucleosome core particle. Our simulations reveal that the CTD preferentially interacts with DNA at the entry/exit near the enzymes docking site, exhibiting a variety of different binding modes. In specific cases, the CTD participates to the promotion of DNA unwrapping and promotes H3K27 accessibility to SIRT6s active site, suggesting a pivotal role of this domain for H3K27 deacetylation. This work provides new structural insights into the binding process of the full-length SIRT6 to a nucleosome core particle, shedding light on the conformational behavior and functional role of its CTD. It constitutes an important step towards the understanding of SIRT6 deacetylation mechanisms and specificity.

biochemistry↗

bioRxiv2023

Binding to nucleosome poises SIRT6 for histone H3 de-acetylation

Sirtuin 6 (SIRT6) is an NAD+ dependent histone H3 deacetylase that is prominently found associated with chromatin, attenuates transcriptionally active promoters and regulates DNA repair, metabolic homeostasis and lifespan. Unlike other sirtuins, it has low affinity to free histone tails but demonstrates strong binding to nucleosomes. It is poorly understood how SIRT6 docking on nucleosomes stimulates its histone deacetylation activity. Here we present the structure of human SIRT6 bound to a nucleosome determined by cryogenic electron microscopy. The zinc finger domain of SIRT6 associates tightly with the acidic patch of the nucleosome through multiple arginine anchors. The Rossmann fold domain binds to the terminus of the looser DNA half of the nucleosome, detaching two turns of the DNA from the histone octamer and placing the NAD+ binding pocket close to the DNA exit site. This domain shows flexibility with respect to the fixed zinc finger and moves with, but also relative to, the unwrapped DNA terminus. We apply molecular dynamics simulations of the histone tails in the nucleosome to show that in this mode of interaction, the active site of SIRT6 is perfectly poised to catalyze deacetylation of the H3 histone tail and that the partial unwrapping of the DNA allows even lysines close to the H3 core to reach the enzyme.

molecular biology↗

bioRxiv2018

The CryoEM Structure of the Ribosome Maturation Factor Rea1

The biogenesis of the 60S ribosomal subunit is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an -helical bundle of AAA2 as a major ATPase activity regulator. The -helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain of Rea1 on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

molecular biology↗