Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

Gutierrez, Craig; Chemmama, Ilan E.; Mao, Haibin; Yu, Clinton; Echeverria, Ignacia; Block, Sarah; Rychnovsky, Scott D.; Zheng, Ning; Šali, Andrej; Huang, Lan

doi:10.1073/pnas.1915542117

Cited by 61 publications

(89 citation statements)

References 61 publications

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“…Our scoring function consisted of the sum of four component restraints. 1) The chemical cross-link restraint utilized the identified chemical cross-links to construct a Bayesian scoring function (Gutierrez et al, 2020) that restrained distances between cross-linked residues. Restraint distances of 30Å for LC-SDA and DSS, and 25Å for EDC were used.…”

Section: Methodsmentioning

confidence: 99%

Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain

Rafiei

Lee

Crowder

et al. 2021

Preprint

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Doublecortin (DCX) is a microtubule (MT) associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX remain poorly resolved. Here, we describe the molecular architecture of the DCX-MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-EM and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving both the C-terminal structured domain and the C-tails, in a conformation that favors an open, domain-swapped state. The networked state can accommodate multiple different attachment points on the MT lattice, all of which orient the C-tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in the C-tail, our study shows that lattice-driven self-assembly is an important property of DCX.

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Section: Methodsmentioning

confidence: 99%

Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain

Rafiei

Lee

Crowder

et al. 2021

Preprint

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“…One approach to overcome the limitations of individual methods is integrative structure modeling 27–32,76–79 . Integrative modeling aims to simultaneously use all available information from any method, including from varied experiments (e.g., EM and chemical cross‐linking), physical theories (e.g., molecular mechanics force field), statistical analyses (e.g., statistical potentials), and prior models (e.g., atomic structures of complex subunits).…”

Section: Discussionmentioning

confidence: 99%

“…The architecture of the yeast exocyst complex was determined using the integrative structure modeling approach described previously 27–32,76–79 . All available structural information on the exocyst complex was used for computational analyses (Table S2, Figure 3, and Section 4).…”

Section: Methodsmentioning

confidence: 99%

Integrative structure and function of the yeast exocyst complex

et al. 2020

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Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative-stain electron microscopy (EM) at~16 Å resolution, 434 disuccinimidyl suberate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride cross-links from chemical-crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo-EM structure, cross-links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo-EM structure, except for Sec5. While not observed in the cryo-EM map, the integrative model localizes the N-terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low-resolution Abbreviations: CATCHR, complexes associated with tethering containing helical rods; CXMS, chemical-crosslinking mass spectrometry; DSS, disuccinimidyl suberate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EM, electron microscopy; GMM, Gaussian mixture model; IMP, integrative modeling platform; LC/MS, liquid chromatography and mass spectrometry; MTC, multisubunit tethering complex; PH domain, Pleckstrin homology; PMI, python modeling interface; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation; SNARE, soluble NSF attachment protein receptor. Sai J. Ganesan and Michael J. Feyder authors contributed equally to this work.

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“…Distinct, non‐overlapping clusters of solutions, as observed in Figure 3a in the XLs_sum parameter (total crosslink score), can indicate insufficient sampling; there are two distinct sets of solutions with different sets of crosslinks satisfied. In comparison, analysis of the more extensive sampling protocol from analysis_extensive (Figure 3b) shows a continuous distribution, indicating a more complete sampling 23,25,26 …”

Section: Integrative Modeling Of the Rna Polymerase II Stalkmentioning

confidence: 98%

Using Integrative Modeling Platform to compute, validate, and archive a model of a protein complex structure

et al. 2020

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Biology is advanced by producing structural models of biological systems, such as protein complexes. Some systems are recalcitrant to traditional structure determination methods. In such cases, it may still be possible to produce useful models by integrative structure determination that depends on simultaneous use of multiple types of data. An ensemble of models that are sufficiently consistent with the data is produced by a structural sampling method guided by a data-dependent scoring function. The variation in the ensemble of models quantified the uncertainty of the structure, generally resulting from the uncertainty in the input information and actual structural heterogeneity in the samples used to produce the data. Here, we describe how to generate, assess, and interpret ensembles of integrative structural models using our open source Integrative Modeling Platform program (https://integrativemodeling.org).

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Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

Cited by 61 publications

References 61 publications

Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain

Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain

Integrative structure and function of the yeast exocyst complex

Using Integrative Modeling Platform to compute, validate, and archive a model of a protein complex structure

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