Molecular architecture of nucleosome remodeling and deacetylase sub‐complexes by integrative structure determination

Arvindekar, Shreyas; Jackman, Matthew J.; Low, Jason K. K.; Landsberg, Michael J.; Mackay, Joel P.; Viswanath, Shruthi

doi:10.1002/pro.4387

Cited by 9 publications

(16 citation statements)

References 64 publications

(299 reference statements)

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“…Typically, studies on intrinsically disordered proteins that undergo phase separation involve small, truncated regions of proteins that are more amenable to biophysical studies. Previous studies of MBD2 and MBD3 have characterized truncated forms of MBD2 and MBD3, but still suffered from low yields due to degradation and aggregation (30,31,56). We have also encountered extensive protein degradation but have adapted a protocol effective for intrinsically disordered proteins and optimized it for MBD2 and MBD3 (32,57).…”

Section: Inducing Mbd2 and Mbd3 Llps In Vitro And In Silicomentioning

confidence: 99%

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

Maurici,

Phan,

Henty-Ridilla

et al. 2024

Preprint

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Chromatin organization controls DNA's accessibility to regulatory factors to influence gene expression. Heterochromatin, or transcriptionally silent chromatin enriched in methylated DNA and methylated histone tails, self-assembles through multivalent interactions with its associated proteins into a condensed, but dynamic state. Liquid-liquid phase separation (LLPS) of key heterochromatin regulators, such as heterochromatin protein 1 (HP1), plays an essential role in heterochromatin assembly and function. Methyl-CpG-binding protein 2 (MeCP2), the most studied member of the methyl-CpG-binding domain (MBD) family of proteins, has been recently shown to undergo LLPS in the absence and presence of methylated DNA. These studies provide a new mechanistic framework for understanding the role of methylated DNA and its readers in heterochromatin formation. However, the details of the molecular interactions by which other MBD family members undergo LLPS to mediate genome organization and transcriptional regulation are not fully understood. Here, we focus on two MBD proteins, MBD2 and MBD3, that have distinct but interdependent roles in gene regulation. Using an integrated computational and experimental approach, we uncover the homotypic and heterotypic interactions governing MBD2 and MBD3 phase separation and DNA's influence on this process. We show that despite sharing the highest sequence identity and structural homology among all the MBD protein family members, MBD2 and MBD3 exhibit differing residue patterns resulting in distinct phase separation mechanisms. Understanding the molecular underpinnings of MBD protein condensation offers insights into the higher-order, LLPS-mediated organization of heterochromatin.

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Section: Inducing Mbd2 and Mbd3 Llps In Vitro And In Silicomentioning

confidence: 99%

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

Maurici,

Phan,

Henty-Ridilla

et al. 2024

Preprint

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“…About 24866 good-scoring models were selected for further analysis (see Methods, Stage 4 for details). These models were clustered based on structural similarity and the precision of the clusters was estimated (Arvindekar et al, 2022;Saltzberg et al, 2021;Viswanath, Chemmama, et al, 2017) (Fig. S2).…”

Section: Summary Of the Integrative Modeling Workflowmentioning

confidence: 99%

“…We employed Gibbs sampling Replica Exchange Monte Carlo sampling (Arvindekar et al, 2022;Saltzberg et al, 2021;Viswanath, Chemmama, et al, 2017). The positions of the rigid bodies and flexible beads were sampled as in previous protocols, with a few customizations.…”

Section: Stage 3: Structural Sampling To Produce An Ensemble Of Struc...mentioning

confidence: 99%

“…The sampled models were analyzed to assess sampling exhaustiveness and estimate the precision of the structure, its consistency with input data and consistency with data not used in modeling. We based our analysis on the protocols published earlier (Arvindekar et al, 2022;Saltzberg et al, 2021;Viswanath, Chemmama, et al, 2017;Webb et al, 2018).…”

Section: Stage 4: Analyzing and Validating The Ensemble Of Structuresmentioning

confidence: 99%

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The molecular architecture of the desmosomal outer dense plaque by integrative structural modeling

Pasani

Menon

Viswanath

2023

Preprint

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Desmosomes are protein assemblies that mediate cell-cell adhesion and are prevalent in tissues under mechanical stress, such as heart and epithelial tissues. However, their detailed structural characterization is not yet available. Here, we characterized the molecular architecture of the desmosomal outer dense plaque (ODP) using Bayesian integrative structural modeling via IMP (Integrative Modeling Platform; https://integrativemodeling.org). We integrated information from X-ray crystallography, electron cryo-tomography, immuno-electron microscopy, yeast two-hybrid experiments, co-immunoprecipitation, in vitro overlay, in vivo co-localization assays, in-silico sequence-based predictions of transmembrane and disordered regions, homology modeling, and stereochemistry information to generate an integrative structure of the ODP. The structure was validated by additional information from biochemical assays that was not used in modeling. The ODP resembles a densely packed cylinder with two layers: a PKP layer and a PG layer; the desmosomal cadherins and PKP span the two layers. We identified previously unknown protein-protein interfaces between DP and Dsc, DP and PG, and PKP and the desmosomal cadherins. The integrative structure sheds light on the function of disordered regions, such as the N-terminus of PKP (N-PKP) and C-terminus of PG in desmosome assembly. In our structure, N-PKP interacts with several proteins in the PG layer, alluding to its importance in desmosome assembly, and implying that it is not merely a structural filler as previously posited. Further, we identified the structural basis for defective cell-cell adhesion in Naxos disease, Carvajal Syndrome, Skin Fragility/Woolly Hair Syndrome, and cancers via mapping of disease-related mutations on the structure. Finally, we point to features of the structure that could confer resilience to mechanical stress, such as the PG-DP interaction and the embedding of cadherins amidst the other proteins. Taken together, we contribute the most complete and robustly validated model of the desmosomal ODP so far, providing mechanistic insight into the function and assembly of desmosomes in normal and disease states.

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“…Third, structural sampling proceeds using the Markov Chain Monte Carlo algorithm. Finally, sampled models are analyzed and validated using previously established protocols, which include assessments of sampling exhaustiveness, clustering, and estimation of model precision (Viswanath et al 2017;Pasani and Viswanath 2021;Saltzberg et al 2021;Arvindekar et al 2022;Ullanat, Kasukurthi and Viswanath 2022).…”

Section: Introductionmentioning

confidence: 99%

Optimizing representations for integrative structural modeling using Bayesian model selection

Arvindekar,

Pathak,

Majila

et al. 2023

Preprint

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MotivationIntegrative structural modeling combines data from experiments, physical principles, statistics of previous structures, and prior models to obtain structures of macromolecular assemblies that are challenging to characterize experimentally. The choice of model representation is a key decision in integrative modeling, as it dictates the accuracy of scoring, efficiency of sampling, and resolution of analysis. But currently, the choice is usually madead hoc, manually.ResultsHere, we report NestOR (Nested Sampling forOptimizingRepresentation), a fully automated, statistically rigorous method based on Bayesian model selection to identify the optimal coarse-grained representation for a given integrative modeling setup. Given an integrative modeling setup, it determines the optimal representations from given candidate representations based on their model evidence and sampling efficiency. The performance of NestOR was evaluated on a benchmark of four macromolecular assemblies.AvailabilityNestOR is implemented in the Integrative Modeling Platform (https://integrativemodeling.org) and is available athttps://github.com/isblab/nestor.Data for the benchmark is athttps://www.doi.org/10.5281/zenodo.10360718.Supplementary Information is available online.

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Molecular architecture of nucleosome remodeling and deacetylase sub‐complexes by integrative structure determination

Cited by 9 publications

References 64 publications

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

The molecular architecture of the desmosomal outer dense plaque by integrative structural modeling

Optimizing representations for integrative structural modeling using Bayesian model selection

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