National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01-GM07391
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
T32-GM008275
United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
F31-HL146077
United States
Citation
Journal: Nat Commun / Year: 2022 Title: Molecular mechanism of Arp2/3 complex inhibition by Arpin. Authors: Fred E Fregoso / Trevor van Eeuwen / Gleb Simanov / Grzegorz Rebowski / Malgorzata Boczkowska / Austin Zimmet / Alexis M Gautreau / Roberto Dominguez / Abstract: Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to ...Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin's mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.
History
Deposition
Aug 9, 2020
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Header (metadata) release
Feb 9, 2022
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Map release
Feb 9, 2022
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Update
May 15, 2024
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Current status
May 15, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Instrument: LEICA EM CPC Details: Grids were manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged using a Leica EM CPC manual plunger..
Details
This sample was monodisperse.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Details
Data were collected in super-resolution mode with an illuminated area of 1.01 um, nominal dose of 40 e-/A^2, a dose rate of 4.87 e-/s/pixel, and 2 or 5 exposures per hole by image shift.
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 9661 / Average exposure time: 2.6 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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