A microtubule crosslinking protocol for integrative structural modeling activities

Rafiei, Atefeh; Schriemer, David C.

doi:10.1016/j.ab.2019.113416

Cited by 6 publications

(5 citation statements)

References 37 publications

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“…That is, conformations not representative of the structural ensemble can be selected based simply on higher reaction rates. Therefore, we used a crosslinker method optimized for MT interactions involving the heterobifunctional reagent LC-SDA that has been demonstrated to minimize this effect (Ziemianowicz et al, 2018, Rafiei and Schriemer, 2019). The first coupling is to accessible nucleophiles through a conventional NHS ester and the second coupling to any surface-accessible residue through laser-initiated carbene chemistry.…”

Section: Resultsmentioning

confidence: 99%

“…Raw LC-MS/MS data were imported into CRIMP v2 (the crosslinking plugin in the Mass Spec Studio, www.msstudio.ca) (Sarpe et al, 2016) along with the Fasta files of the Tubulin isoforms identified previously (TB-α-1A, TB-α-1B, TB-α-1C, TB-α-1D, TB-α-4A, and TB-β, TB-β-2B, TB-β-4A, TB-β-4B, TB-β-3, TB-β-5) (Rafiei and Schriemer, 2019). The minor impurities in the recombinantly purified DCX did not have an impact on crosslinking results.…”

Section: Methodsmentioning

confidence: 99%

“…Further, how DCX is functionally altered by phosphorylation (Tanaka et al, 2004, Graham et al, 2004, Shmueli et al, 2006) and engages coregulatory proteins like Neurabin II (Tsukada et al, 2005) and kinesin-3 motor protein (Liu et al, 2012, Lipka et al, 2016) requires a complete structure of the full length protein on the MT lattice. Here, we develop a unifying model of the DCX-MT interaction through an integrative structure-building approach, using data from an improved crosslinking mass spectrometry (XL-MS) method (Ziemianowicz et al, 2018, Rafiei and Schriemer, 2019), together with available cryo-EM and X-ray crystallographic structures. Our results support a DCX-MT interaction model in which NDC binds to MTs at the junction of the four α/β-tubulin dimers, and induces DCX self-association through its CDC and C-tail domains, creating an extended structure capable of capturing different lateral and longitudinal MT interactions.…”

Section: Introductionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Rafiei

Lee

Crowder

et al. 2021

Preprint

Self Cite

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show abstract

“…Succinimidyl 6-(4,4′-azipentanamido) hexanoate cross-linked Lys 163 from alpha tubulin with Lys 163 from beta tubulin, and it cross-linked Lys 154 to Glu 158 in beta tubulin. 28 Incubation of Tau with microtubules in the presence of bis(sulfosuccinimidyl)suberate resulted in covalent binding of Tau to Lys 336 and Lys 338 of alpha tubulin. 29 Thus, Lys 163 and Lys 336 in alpha tubulin appear to be particularly prone to cross-linking.…”

Section: Discussionmentioning

confidence: 99%

“…Reaction with 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide cross-linked Asp 218 and Lys 163 in alpha tubulin. Succinimidyl 6-(4,4′-azipentanamido) hexanoate cross-linked Lys 163 from alpha tubulin with Lys 163 from beta tubulin, and it cross-linked Lys 154 to Glu 158 in beta tubulin . Incubation of Tau with microtubules in the presence of bis(sulfosuccinimidyl)suberate resulted in covalent binding of Tau to Lys 336 and Lys 338 of alpha tubulin .…”

Section: Discussionmentioning

confidence: 99%

Organophosphorus Pesticides Promote Protein Cross-Linking

Schopfer

Onder

Lockridge

2022

Chem. Res. Toxicol.

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Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 μg of MAP-rich tubulin with 100 μM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.

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A Crosslinking Mass Spectrometry Protocol for the Structural Analysis of Microtubule-Associated Proteins

Rafiei

Schriemer

2022

Methods in Molecular Biology

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A microtubule crosslinking protocol for integrative structural modeling activities

Cited by 6 publications

References 37 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

Organophosphorus Pesticides Promote Protein Cross-Linking

A Crosslinking Mass Spectrometry Protocol for the Structural Analysis of Microtubule-Associated Proteins

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