Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome Complex

Tosi, Alessandro; Haas, Caroline; Herzog, Franz; Gilmozzi, Andrea; Berninghausen, Otto; Ungewickell, Charlotte; Gerhold, Christian-Benedikt; Lakomek, K.; Aebersold, Ruedi; Beckmann, Roland; Hopfner, Karl‐Peter

doi:10.1016/j.cell.2013.08.016

Cited by 207 publications

(340 citation statements)

References 75 publications

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“…CXMS has emerged as an important structural technique to identify potential subunit interaction interfaces in the context of the intact, fully assembled macromolecular assembly. [41][42][43] Using this novel approach, we were able to comprehensively map the subunit interaction interfaces of a reconstituted minimal Atg1 pentameric complex from the model organism S. cerevisiae (Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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(2015) Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms, Autophagy, 11:6, 891-905, DOI: 10.1080/15548627.2015 The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1 [CTD]) and Atg13 (Atg13 [CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1 [CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

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Section: Discussionmentioning

confidence: 99%

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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“…Tosi et al (16) built a model from cryo-EM data of the yeast INO80 complex to a resolution of ϳ17 Å. The model (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A number of low resolution models of these remodeling complexes (23-50 Å) derived from single-particle electron microscopy and cryo-EM data have been reported (9 -17); these models reveal some diversity in the overall shape of each complex, but a common feature is the integral nature of the remodeling subunit. For example, in the recent INO80 and SWR1 structures, the Ino80 and Swr1 ATPases lie at the center of the corresponding complex and are decorated with sets of regulatory subunits (14,16).…”

mentioning

confidence: 99%

CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex

Low

Webb

Silva

et al. 2016

Journal of Biological Chemistry

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Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ϳ10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.Nucleosomes effectively act as a roadblock to all aspects of genome biology. ATP-dependent chromatin remodeling enzymes solve this problem by using ATP-derived energy to alter the positions, occupancy and composition of nucleosomes. All remodelers possess a highly related ATPase motor domain from the helicase family and are classified into four subfamilies (INO80, ISWI, SWR1, and CHD) based on sequence similarity (1). Each subfamily is represented in nearly all eukaryotes, suggesting that they catalyze different remodeling events. For example, ISWI proteins reposition (or slide) nucleosomes to create regularly spaced arrays; this periodic organization is a key characteristic of DNA at the start of genes (2). SWR1 and INO80 enzymes have opposing roles in histone variant dynamics; the former incorporates these histone variants (e.g. H2A.Z), whereas the latter removes them. These variants set up specific chromatin structures that modulate transcription and replication, although the roles of many variants are still under debate (3). Fundamentally, these remodeling enzymes all alter the accessibility of DNA to other DNA-binding factors and thereby broadly underpin genome biology.Remodelers frequently act in the context of large multisubunit complexes, and in general, the "mixing and matching" of complex composition can generate complexes with varying activities; the human ISWI protein Snf2h for instance has been identified in six distinct complexes (4). Likewise, the accessory subunits can also modulate remodeler activity. For example, the paralogous methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) subunits of the nucleosome remodeling and deacetylase (NuRD) 6 complex are mutually exclusive (5); MBD2 recognizes 5-methylcytosine-modified DNA, whereas MBD3 instead binds to 5-hydroxymethylated DNA (6, 7). Unsurprisingly, it has been observed tha...

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“…Recent crosslinking studies have targeted several families of remodeler enzymes. For example, the Aebersold group contributed to two hybrid structural biology projects focusing on the remodelers INO80 [91] and Swi2/Snf2-related 1 (SWR1) [92]. In both cases, XL-MS data aided in positioning individual subunits into EM maps.…”

Section: Ribosomes and Associated Proteinsmentioning

confidence: 99%

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

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In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

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Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome Complex

Cited by 207 publications

References 75 publications

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

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