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- PDB-8ssl: Isobutyryl-CoA mutase fused Q341A in the presence of GTP -

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Basic information

Entry
Database: PDB / ID: 8ssl
TitleIsobutyryl-CoA mutase fused Q341A in the presence of GTP
ComponentsFused isobutyryl-CoA mutase
KeywordsISOMERASE / supramolecular complex / b12-binding / g-protein chaperone
Function / homology
Function and homology information


isobutyryl-CoA mutase / pivalyl-CoA mutase activity / isobutyryl-CoA mutase activity / methylmalonyl-CoA mutase activity / acyl-CoA metabolic process / cobalamin binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / GTPase activity / GTP binding / magnesium ion binding
Similarity search - Function
Fused isobutyryl-CoA mutase / Methylmalonyl Co-A mutase-associated GTPase MeaB / Methylmalonyl-CoA mutase, alpha chain, catalytic / Methylmalonyl-CoA mutase, alpha/beta chain, catalytic / Methylmalonyl-CoA mutase / Methylmalonyl-CoA mutase, C-terminal / Cobalamin (vitamin B12)-dependent enzyme, catalytic / B12 binding domain / Cobalamin-binding domain superfamily / B12-binding domain profile. ...Fused isobutyryl-CoA mutase / Methylmalonyl Co-A mutase-associated GTPase MeaB / Methylmalonyl-CoA mutase, alpha chain, catalytic / Methylmalonyl-CoA mutase, alpha/beta chain, catalytic / Methylmalonyl-CoA mutase / Methylmalonyl-CoA mutase, C-terminal / Cobalamin (vitamin B12)-dependent enzyme, catalytic / B12 binding domain / Cobalamin-binding domain superfamily / B12-binding domain profile. / Cobalamin (vitamin B12)-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / Fused isobutyryl-CoA mutase
Similarity search - Component
Biological speciesCupriavidus metallidurans CH34 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsVaccaro, F.A. / Drennan, C.L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31 GM131648 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM126982 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Biol Chem / Year: 2023
Title: Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.
Authors: Francesca A Vaccaro / Daphne A Faber / Gisele A Andree / David A Born / Gyunghoon Kang / Dallas R Fonseca / Marco Jost / Catherine L Drennan /
Abstract: G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the ...G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(β,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.
History
DepositionMay 8, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Fused isobutyryl-CoA mutase
B: Fused isobutyryl-CoA mutase
C: Fused isobutyryl-CoA mutase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)369,9657
Polymers368,6123
Non-polymers1,3544
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Negative stain electron microscopy was performed.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Fused isobutyryl-CoA mutase


Mass: 122870.523 Da / Num. of mol.: 3 / Mutation: Q341A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cupriavidus metallidurans CH34 (bacteria)
Gene: icmF, sbm, Rmet_0210 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q1LRY0, isobutyryl-CoA mutase, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#2: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Supramolecular complex of isobutyryl-CoA mutase fused / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Cupriavidus metallidurans CH34 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Details: 20 mM HEPES pH 8, 50 mM NaCl, 500 mM GTP, 500 mM MgCl2
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281.15 K / Details: Blot for 3s

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3100 nm / Nominal defocus min: 1200 nm
Image recordingAverage exposure time: 3.99 sec. / Electron dose: 58.71 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 673

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 180271
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59335 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 4XC6

4xc6


Pdb chain-ID: B / Accession code: 4XC6 / Chain residue range: 22-1093
Details: Docked the model as two different fragments: resi 22-442 and resi 443-1093
Pdb chain residue range: 22-1093 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325115
ELECTRON MICROSCOPYf_angle_d0.51334025
ELECTRON MICROSCOPYf_dihedral_angle_d14.0789206
ELECTRON MICROSCOPYf_chiral_restr0.0393789
ELECTRON MICROSCOPYf_plane_restr0.0044527

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