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Molecular architecture of the augmin complex

ABSTRACT: Haus DSSO CX-MS analysis to validate Cyro-EM analysis of Haus multimer

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrew DeMarco

LAB HEAD: Mark C. Hall

PROVIDER: PXD031411 | Pride | 2022-06-29

REPOSITORIES: Pride

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			Action	DRS
	100-DSSO-insolution.mzML	Mzml
	100-DSSO-insolution.raw	Raw
	1000-DSSO-ingel.mzML	Mzml
	1000-DSSO-ingel.raw	Raw
	250-DSSO-ingel.mzML	Mzml

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Analysis of conformational changes induced by binding of allosteric nanobodies to a bacterial LRRK2 homologue by CX-MS

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Conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

Project description:Crosslinking mass spectrometry study of an important adapter protein complex at the Golgi AP-3. We performed crosslinking mass spectrometry study using crosslinker DSSO and analyzed the digested peptides by LC-MS. The crosslinking study provided important information the subunit connectivity of AP-3, which facilitated the structural analysis of AP-3 by cyro-EM.

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Molecular architecture of the augmin complex.

Project description:Accurate segregation of chromosomes during mitosis depends on the correct assembly of the mitotic spindle, a bipolar structure composed mainly of microtubules. The augmin complex, or homologous to augmin subunits (HAUS) complex, is an eight-subunit protein complex required for building robust mitotic spindles in metazoa. Augmin increases microtubule density within the spindle by recruiting the γ-tubulin ring complex (γ-TuRC) to pre-existing microtubules and nucleating branching microtubules. Here, we elucidate the molecular architecture of augmin by single particle cryo-electron microscopy (cryo-EM), computational methods, and crosslinking mass spectrometry (CLMS). Augmin's highly flexible structure contains a V-shaped head and a filamentous tail, with the head existing in either extended or contracted conformational states. Our work highlights how cryo-EM, complemented by computational advances and CLMS, can elucidate the structure of a challenging protein complex and provides insights into the function of augmin in mediating microtubule branching nucleation.

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The augmin complex architecture reveals structural insights into microtubule branching.

Project description:In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.

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DSSO,Xlink,LC-MSMS - DSSO as linker, study of protein-protein interaction by LC-MSMS

Project description:DSSO as linker, study of protein-protein interaction by LC-MSMS

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study of protein-protein interaction by LC-MSMS(28)

Project description:DSSO as linker, study of protein-protein interaction by LC-MSMS

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DSSO,Xlink,LC-MSMS - DSSO as linker, study of protein-protein interaction by LC-MSMS

Project description:DSSO as linker, study of protein-protein interaction by LC-MSMS

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study of protein-protein interaction by LC-MSMS

Project description:DSSO as linker, study of protein-protein interaction by LC-MSMS

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Reconstitution of the augmin complex provides insights into its architecture and function.

Project description:Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin's structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures.

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Project description:Gap junctions are arrays of intercellular channels formed by two hemichannels docked end-to-end. Each hemichannel is a hexamer containing the same or different connexin (Cx) isoform. While homomeric Cxs forms have been largely described functionally and structurally, lesser is known about heteromeric Cx channels. To unveil properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even for only combining two Cx isoforms. We engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments. For Cx26-HA/Cx30 or Cx26/Cx30-HA heteromeric channels, the Fab-HA binding distribution followed a binomial distribution with a maximum of 3 Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements which can be derived from the law of total probabilities. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30.

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