Ian Cooney

All Publications

• Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway NATURE COMMUNICATIONS Cooney, I., Schubert, H. L., Cedeno, K., Fisher, O. N., Carson, R., Price, J. C., Hill, C. P., Shen, P. S. 2024; 15 (1): 7505

  Abstract

  The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding.

  View details for DOI 10.1038/s41467-024-51835-3

  View details for Web of Science ID 001377069900001

  View details for PubMedID 39209885

  View details for PubMedCentralID PMC11362554

• Lysate-to-grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging BIO-PROTOCOL Cooney, I., Mack, D. C., Ferrell, A. J., Stewart, M. G., Wang, S., Donelick, H. M., Tamayo-Jaramillo, D., Greer, D. L., Zhu, D., Li, W., Shen, P. S. 2023; 13 (2)

  Abstract

  Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow. Here, we present a protocol using budding yeast ( S. cerevisiae ), in which a tractable immunoprecipitation tag (3xFLAG) is appended at the endogenous locus of a gene of interest (GOI). The modified gene is expressed under its endogenous promoter, and cells are grown and harvested using standard procedures. Our protocol describes the steps in which the tagged proteins and their associated complexes are isolated within three hours of thawing cell lysates, after which the recovered proteins are used directly for cryo-EM specimen preparation. The prioritization of speed maximizes the ability to recover intact, scarce complexes. The protocol is generalizable to soluble yeast proteins that tolerate C-terminal epitope tags. Graphical abstract Overview of lysate-to-grid workflow. Yeast cells are transformed to express a tractable tag on a gene of interest. Following cell culture and lysis, particles of interest are rapidly isolated by co-immunoprecipitation and prepared for cryo-EM imaging (created with BioRender.com).

  View details for DOI 10.21769/BioProtoc.4596

  View details for Web of Science ID 000924224000007

  View details for PubMedID 36789166

  View details for PubMedCentralID PMC9901474

• Active conformation of the p97-p47 unfoldase complex. Nature communications Xu, Y., Han, H., Cooney, I., Guo, Y., Moran, N. G., Zuniga, N. R., Price, J. C., Hill, C. P., Shen, P. S. 2022; 13 (1): 2640

  Abstract

  The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a "hand-over-hand" mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification.

  View details for DOI 10.1038/s41467-022-30318-3

  View details for PubMedID 35552390

  View details for PubMedCentralID PMC9098461

• Structure of the Cdc48 segregase in the act of unfolding an authentic substrate SCIENCE Cooney, I., Han, H., Stewart, M. G., Carson, R. H., Hansen, D. T., Iwasa, J. H., Price, J. C., Hill, C. P., Shen, P. S. 2019; 365 (6452): 502-+

  Abstract

  The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.7-angstrom-resolution structure of Cdc48 in complex with an adaptor protein and a native substrate. Cdc48 engages substrate by adopting a helical configuration of substrate-binding residues that extends through the central pore of both of the ATPase rings. These findings indicate a unified hand-over-hand mechanism of protein translocation by Cdc48 and other AAA+ ATPases.

  View details for DOI 10.1126/science.aax0486

  View details for Web of Science ID 000485768400049

  View details for PubMedID 31249134

  View details for PubMedCentralID PMC7362759