Input data, modeling scripts, and integrative models of the γTuSC-Spc110 complex with Spc110 in monomer, dimer, and tetramer forms. For more information about how to reproduce this modeling, see https://salilab.org/gtuscSpc110 or the README file.
Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae , γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.
Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.
Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.
Significance Arp2/3 complex is a macromolecular machine that nucleates branched actin filaments in response to cellular signals. WASP family proteins regulate the nucleation activity of Arp2/3 complex, providing a way for cells to assemble branched actin filament networks with the spatiotemporal precision required to orchestrate cellular processes such as cellular motility and endocytosis. Despite its importance, how WASP activates filament nucleation by Arp2/3 complex is still unknown, largely because it has been uncertain how WASP binds to Arp2/3 complex. Here we use a combination of biophysical, biochemical, and computational methods to map the binding of WASP to the complex. These results have important implications for understanding how cells regulate the assembly of actin filaments.
The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.
