Journal: Nat Struct Mol Biol / Year: 2023 Title: Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments. Authors: Wout Oosterheert / Florian E C Blanc / Ankit Roy / Alexander Belyy / Micaela Boiero Sanders / Oliver Hofnagel / Gerhard Hummer / Peter Bieling / Stefan Raunser / Abstract: The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release ...The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases P through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why P escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated P release and filaments with drastically shortened ADP-P caps. Our results provide the molecular basis for P release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.
Entire : Actin filament harboring the N111S mutation.
Entire
Name: Actin filament harboring the N111S mutation.
Components
Complex: Actin filament harboring the N111S mutation.
Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
Ligand: ADENOSINE-5'-DIPHOSPHATE
Ligand: MAGNESIUM ION
Ligand: water
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Supramolecule #1: Actin filament harboring the N111S mutation.
Supramolecule
Name: Actin filament harboring the N111S mutation. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Beta-actin was expressed as fusion protein, with thymosin beta4 and a deca-His-tag fused to the actin C-terminus. During the purification, thymosin beta-4 and the deca-His-tag were removed. ...Details: Beta-actin was expressed as fusion protein, with thymosin beta4 and a deca-His-tag fused to the actin C-terminus. During the purification, thymosin beta-4 and the deca-His-tag were removed. Actin was purified as monomer from insect cells. It was then polymerized into a filament in vitro.
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) Details: The final refinement was performed from local searches in RELION. Number images used: 1756928
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.4) Details: First 3D refinement was performed in helical SPHIRE with meridien alpha, which imposes helical restraints to limit particle shifts to the helical rise to prevent particle duplication, but ...Details: First 3D refinement was performed in helical SPHIRE with meridien alpha, which imposes helical restraints to limit particle shifts to the helical rise to prevent particle duplication, but does not apply helical symmetry
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final 3D classification
Number classes: 8 / Software - Name: RELION (ver. 3.1.0) Details: 3D classification without image alignment was performed in RELION to remove particles that did not contain high-resolution information.
Chain - Source name: PDB / Chain - Initial model type: experimental model
Details
chain C of pdb 8A2T (including all water molecules) was fit in the central actin subunit of the density map. After substitution of all alpha-actin specific amino-acids to the corresponding beta-actin residues, introducing the N111S mutation, and further manual model building in Coot, the resulting model was fitted in four more actin subunits (chains A, B, D, E) in the density map. The filament was modeled as a pentamer to capture the full interaction interface of the central subunit with its four neighboring subunits. All water molecules were first manually built, inspected and adjusted in the central subunit, and were then copied to the other chains with non-crystallographic symmetry (NCS). Because the local resolution was worse at the periphery of the reconstruction, we removed water molecules that displayed poor corresponding cryo-EM density in the non-central actin chains. The model was refined in Phenix real-space refine with NCS restraints but without Ramachandran and rotamer restraints.
Refinement
Space: REAL / Protocol: FLEXIBLE FIT
Output model
PDB-8oid: Cryo-EM structure of ADP-bound, filamentous beta-actin harboring the N111S mutation
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