Structure of the ISW1a complex bound to the dinucleosome

Li, Lifei; Chen, Kangjing; Sia, Youyang; Hu, Pengjing; Ye, Youpi; Chen, Zhucheng

doi:10.1101/2023.01.02.522444

Cited by 4 publications

(3 citation statements)

References 65 publications

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“…ISWI has two known DNA binding domains, its ATPase domain and its HSS domain (Grüne et al, 2003), allowing it to bind two nucleosomes (Bhardwaj et al, 2020; L. Li et al, 2023; Yamada et al, 2011) potentially on neighboring DNA fibers. ISWI-mediated bridges between neighboring fibers would alter mechanical properties of condensates.…”

Section: Resultsmentioning

confidence: 99%

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

Vizjak,

Kamp,

Hepp

et al. 2023

Preprint

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SummaryHow chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using theDrosophilaISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect slidingin vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI’s mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI’s diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a ‘monkey-bar’ model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that ‘monkey-bar’ mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

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Section: Resultsmentioning

confidence: 99%

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

Vizjak,

Kamp,

Hepp

et al. 2023

Preprint

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“…We have mapped this cross-link on a structure of the SANT domain of D. Melanogaster homologue of SMCA5, ISWI (Figure 4C left), but there is no solved structure of SMCA5 that contains the SANT domain. Recently, a structure of S. Cerevisiae ISW1a complexed with two nucleosomes have been solved 34 . The sequence of yeast ISW1a is substantially divergent from fly ISWI (and human SMCA5), but structurally their SANT domains are similar (Supp.…”

Section: Chromatin Remodeling Complexes and Their Interaction With Nu...mentioning

confidence: 99%

Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines

Bakhtina,

Wippel,

Chavez

et al. 2024

Preprint

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Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type-specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.

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“…While the function of this PTM is not known, its location proximal to this critical domain led us to speculate that it might alter ACF remodeling activity. Indeed, structural modeling using an available cyro-EM structure of ISW1 bound to the nucleosome ( 34 ) suggests that this phosphotyrosine is positioned to interact with basic sidechains in the APB motif, thereby affecting its function (Fig. S24).…”

Section: Efficient Protein Transposition Requires Matched Split Intei...mentioning

confidence: 99%

Protein Editing using a Concerted Transposition Reaction

Hua,

Tay,

et al. 2024

Preprint

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Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processesin vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report anin vitrostrategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this ‘protein transposition’ reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multi-subunit chromatin remodeling complex, ACF, where we also show protein transposition can occurin situwithin the cell nucleus. By carrying out a molecular ‘cut and paste’ on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.

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Structure of the ISW1a complex bound to the dinucleosome

Cited by 4 publications

References 65 publications

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines

Protein Editing using a Concerted Transposition Reaction

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