Searched for: person:ricew01
in-biosketch:yes
BRAF oncogenic mutants evade autoinhibition through a common mechanism
Lavoie, Hugo; Jin, Ting; Lajoie, Driss; Decossas, Marion; Gendron, Patrick; Wang, Bing; Filandr, Frantisek; Sahmi, Malha; Hwa Jo, Chang; Weber, Sandra; Arseneault, Geneviève; Tripathy, Sasmita; Beaulieu, Pierre; Schuetz, Doris A; Schriemer, David C; Marinier, Anne; Rice, William J; Maisonneuve, Pierre; Therrien, Marc
Uncontrolled activation of the rat sarcoma (RAS)-extracellular signal-regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving monomeric autoinhibition and activation by dimerization, has been intensely scrutinized, but mechanisms enabling oncogenic mutants to evade regulation remain unclear. By using cryo-electron microscopy, we solved the three-dimensional structures of the three oncogenic BRAF mutant classes, including the common V600E variant. These mutations disrupted wild-type BRAF's autoinhibited state, mediated by interactions between the cysteine-rich domain and kinase domain, thereby shifting the kinase domain into a preactivated conformation. This structural change likely results from helix αC displacement. PLX8394, a BRAF inhibitor that stabilizes helix αC in an inactive conformation, restored the autoinhibited conformation of oncogenic BRAF, explaining the properties of this class of compounds.
PMID: 40440367
ISSN: 1095-9203
CID: 5854792
Substrate translocation and inhibition in human dicarboxylate transporter NaDC3
Li, Yan; Song, Jinmei; Mikusevic, Vedrana; Marden, Jennifer J; Becerril, Alissa; Kuang, Huihui; Wang, Bing; Rice, William J; Mindell, Joseph A; Wang, Da-Neng
The human high-affinity sodium-dicarboxylate cotransporter (NaDC3) imports various substrates into the cell as tricarboxylate acid cycle intermediates, lipid biosynthesis precursors and signaling molecules. Understanding the cellular signaling process and developing inhibitors require knowledge of the structural basis of the dicarboxylate specificity and inhibition mechanism of NaDC3. To this end, we determined the cryo-electron microscopy structures of NaDC3 in various dimers, revealing the protomer in three conformations: outward-open Co, outward-occluded Coo and inward-open Ci. A dicarboxylate is first bound and recognized in Co and how the substrate interacts with NaDC3 in Coo likely helps to further determine the substrate specificity. A phenylalanine from the scaffold domain interacts with the bound dicarboxylate in the Coo state and modulates the kinetic barrier to the transport domain movement. Structural comparison of an inhibitor-bound structure of NaDC3 to that of the sodium-dependent citrate transporter suggests ways for making an inhibitor that is specific for NaDC3.
PMID: 39622972
ISSN: 1545-9985
CID: 5780092
Structural insights into the diversity and DNA cleavage mechanism of Fanzor
Xu, Peiyu; Saito, Makoto; Faure, Guilhem; Maguire, Samantha; Chau-Duy-Tam Vo, Samuel; Wilkinson, Max E; Kuang, Huihui; Wang, Bing; Rice, William J; Macrae, Rhiannon K; Zhang, Feng
Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.
PMCID:11423790
PMID: 39208796
ISSN: 1097-4172
CID: 5719322
Applying 3D ED/MicroED workflows toward the next frontiers
Aragon, Mahira; Bowman, Sarah E J; Chen, Chun Hsing; de la Cruz, M Jason; Decato, Daniel A; Eng, Edward T; Flatt, Kristen M; Gulati, Sahil; Li, Yuchen; Lomba, Charles J; Mercado, Brandon; Miller, Jessalyn; Palatinus, Lukáš; Rice, William J; Waterman, David; Zimanyi, Christina M
We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.
PMCID:11150879
PMID: 38712546
ISSN: 2053-2296
CID: 5664772
Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1
Thomas, Jonathan F; Valencia-Sánchez, Marco Igor; Tamburri, Simone; Gloor, Susan L; Rustichelli, Samantha; GodÃnez-López, Victoria; De Ioannes, Pablo; Lee, Rachel; Abini-Agbomson, Stephen; Gretarsson, Kristjan; Burg, Jonathan M; Hickman, Allison R; Sun, Lu; Gopinath, Saarang; Taylor, Hailey F; Sun, Zu-Wen; Ezell, Ryan J; Vaidya, Anup; Meiners, Matthew J; Cheek, Marcus A; Rice, William J; Svetlov, Vladimir; Nudler, Evgeny; Lu, Chao; Keogh, Michael-Christopher; Pasini, Diego; Armache, Karim-Jean
Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.
PMID: 37556531
ISSN: 2375-2548
CID: 5594932
Measuring the Effect of Ice Thickness and Microscope Configuration on Resolution in Single Particle Cryo-EM
Chua, Eugene Y D; Neselu, Kasahun; Wang, Bing; Rice, William J; Potter, Clinton S; Carragher, Bridget
PMID: 37613233
ISSN: 1435-8115
CID: 5598692
CD19 CAR antigen engagement mechanisms and affinity tuning
He, Changhao; Mansilla-Soto, Jorge; Khanra, Nandish; Hamieh, Mohamad; Bustos, Victor; Paquette, Alice J; Garcia Angus, Andreina; Shore, Derek M; Rice, William J; Khelashvili, George; Sadelain, Michel; Meyerson, Joel R
Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.
PMID: 36867678
ISSN: 2470-9468
CID: 5432422
Structural basis of histone H2A lysine 119 deubiquitination by Polycomb Repressive Deubiquitinase BAP1/ASXL1
Thomas, Jonathan F; Valencia-Sánchez, Marco Igor; Tamburri, Simone; Gloor, Susan L; Rustichelli, Samantha; Godínez-López, Victoria; De Ioannes, Pablo; Lee, Rachel; Abini-Agbomson, Stephen; Gretarsson, Kristjan; Burg, Jonathan M; Hickman, Allison R; Sun, Lu; Gopinath, Saarang; Taylor, Hailey; Meiners, Matthew J; Cheek, Marcus A; Rice, William; Nudler, Evgeny; Lu, Chao; Keogh, Michael-Christopher; Pasini, Diego; Armache, Karim-Jean
UNLABELLED:The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology. ONE SENTENCE SUMMARY/UNASSIGNED:We reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.
PMID: 36865140
ISSN: 2692-8205
CID: 5852342
Measuring the effects of ice thickness on resolution in single particle cryo-EM
Neselu, Kasahun; Wang, Bing; Rice, William J.; Potter, Clinton S.; Carragher, Bridget; Chua, Eugene Y.D.
Ice thickness is a critical parameter in single particle cryo-EM "“ too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50"“150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.
SCOPUS:85147226389
ISSN: 2590-1524
CID: 5424422
Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1
Thomas, Jonathan F.; Valencia-Sanchez, Marco Igor; Tamburri, Simone; Gloor, Susan L.; Rustichelli, Samantha; Godinez-Lopez, Victoria; De Ioannes, Pablo; Lee, Rachel; Abini-Agbomson, Stephen; Gretarsson, Kristjan; Burg, Jonathan M.; Hickman, Allison R.; Sun, Lu; Gopinath, Saarang; Taylor, Hailey F.; Sun, Zu-Wen; Ezell, Ryan J.; Vaidya, Anup; Meiners, Matthew J.; Cheek, Marcus A.; Rice, William J.; Svetlov, Vladimir; Nudler, Evgeny; Lu, Chao; Keogh, Michael-Christopher; Pasini, Diego; Armache, Karim-Jean
ISI:001045489300011
ISSN: 2375-2548
CID: 5852362