A 3.8 Å Resolution Cryo-EM Structure of a Small Protein Bound to a Modular Imaging Scaffold

Y, Liu; Huynh, Duc T.; Yeates, Todd O.

doi:10.1101/505792

Cited by 2 publications

(5 citation statements)

References 39 publications

(44 reference statements)

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“…The cryo-EM structure of E545K ( 38 , 39 ) closely resembles that of E542K with a Cα rmsd value of 1.22 Å ( Fig. 4 D ), significantly lower than that of apo and BYL-719–bound WT or PI3Kα-H1047R (1.73 Å, 1.91 Å, and 2.14 Å, respectively; SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 86%

“…1 D ). This allowed us to build an accurate model for the complex ( 24 , 25 ) ( Fig. 1 E and SI Appendix , Table S1 ), including all domains of p110α and nSH2 and iSH2 domains of p85α and BYL-719.…”

Section: Resultsmentioning

confidence: 99%

“…Under these conditions, the lipid kinase activity of both helical domain mutations was above that of the WT protein, suggesting that the molecular details of inhibitor action are different for the kinase and helical domain mutations. The purified E542K complex ( 32 , 33 ) was analyzed by SDS-PAGE and native gel electrophoresis to verify purity and integrity ( Fig. 3 A and SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The electron density maps and atomic coordinates for BYL-719–bound PI3Kα mutants H1047R, E542K, and E545K have been deposited in the Electron Microscopy Data Bank ( https://www.ebi.ac.uk/pdbe/emdb/ ) under accession numbers EMD-34272 ( 24 ), EMD-34271 ( 32 ), and EMD-34273 ( 38 ) and in the Protein Data Bank ( https://www.rcsb.org ) under accession numbers 8GUB ( 25 ), 8GUA ( 33 ), and 8GUD ( 39 ), respectively. All other study data are included in the article and/or SI Appendix .…”

Section: Data Materials and Software Availabilitymentioning

confidence: 99%

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Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

Liu

Zhou

Hart

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single–amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD–p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Data Materials and Software Availabilitymentioning

confidence: 99%

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Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

Liu

Zhou

Hart

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…The atomic coordinates derived using reconstructions SAT-SB-C2, SAT-SB-C1, SAT-LB-C2, SAT-LB-C1, WT-SB-C2, WT-SB-C1, WT-LB-C2, and WT-LB-C1 have been deposited with the Protein Data Bank [accession numbers 8AFH ( 74 ), 8AFE ( 75 ), 8AJB ( 76 ), 8AHL ( 77 ), 8AFM ( 78 ), 8AFL ( 79 ), 8AIX ( 80 ), 8AIA ( 81 ). The cryo-EM maps of SAT-SB-C2, SAT-SB-C1, SAT-LB-C2, SAT-LB-C1, WT-SB-C2, WT-SB-C1, WT-LB-C2, and WT-LB-C1 have been deposited with the Electron Microscopy Data Bank (accession numbers EMD-15398 ( 82 ), EMD-15395 ( 83 ), EMD-15476 ( 84 ), EMD-15446 ( 85 ), EMD-15402 ( 86 ), EMD-15401 ( 87 ), EMD-15473 ( 88 ), and EMD-15465 ( 89 )]. SI Appendix , Table S3 summarizes details.…”

Section: Data Materials and Software Availabilitymentioning

confidence: 99%

Filament structure and subcellular organization of the bacterial intermediate filament–like protein crescentin

Liu,

van den Ent,

Löwe

2024

Proc. Natl. Acad. Sci. U.S.A.

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The protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus ( vibrioides ). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein’s length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the “stutter.” Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.

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A 3.8 Å Resolution Cryo-EM Structure of a Small Protein Bound to a Modular Imaging Scaffold

Cited by 2 publications

References 39 publications

Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

Filament structure and subcellular organization of the bacterial intermediate filament–like protein crescentin

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