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- PDB-8hf4: Cryo-EM structure of nucleotide-bound ComA at outward-facing stat... -

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Basic information

Entry
Database: PDB / ID: 8hf4
TitleCryo-EM structure of nucleotide-bound ComA at outward-facing state with EC gate closed conformation
ComponentsTransport/processing ATP-binding protein ComA
KeywordsMEMBRANE PROTEIN / TRANSPORT PROTEIN / ABC transport / PCAT
Function / homology
Function and homology information


ABC-type bacteriocin transporter activity / Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate / establishment of competence for transformation / ATPase-coupled lipid transmembrane transporter activity / cysteine-type peptidase activity / transmembrane transport / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / ATP hydrolysis activity / proteolysis / ATP binding / plasma membrane
Similarity search - Function
Peptidase C39, ABC-type bacteriocin transporter / Peptidase family C39 domain profile. / Peptidase C39 family / Peptidase C39, bacteriocin processing / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site ...Peptidase C39, ABC-type bacteriocin transporter / Peptidase family C39 domain profile. / Peptidase C39 family / Peptidase C39, bacteriocin processing / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Transport/processing ATP-binding protein ComA
Similarity search - Component
Biological speciesStreptococcus pneumoniae D39 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYu, L. / Xin, X. / Min, L.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)A-0008412-00-00 Singapore
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of peptide secretion for Quorum sensing by ComA.
Authors: Lin Yu / Xin Xu / Wan-Zhen Chua / Hao Feng / Zheng Ser / Kai Shao / Jian Shi / Yumei Wang / Zongli Li / Radoslaw M Sobota / Lok-To Sham / Min Luo /
Abstract: Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as ...Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA's peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.
History
DepositionNov 9, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 6, 2024Group: Source and taxonomy / Category: em_entity_assembly_naturalsource / entity_src_gen
Item: _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism ..._em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transport/processing ATP-binding protein ComA
B: Transport/processing ATP-binding protein ComA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,9134
Polymers160,8662
Non-polymers1,0462
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transport/processing ATP-binding protein ComA


Mass: 80433.031 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae D39 (bacteria)
Gene: comA, spr0043 / Production host: Escherichia coli (E. coli)
References: UniProt: P59653, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, Translocases; Catalysing the translocation of amino acids and peptides; Linked to the ...References: UniProt: P59653, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate
#2: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
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: ComA / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Streptococcus pneumoniae D39 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 25 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108592 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029050
ELECTRON MICROSCOPYf_angle_d0.48212278
ELECTRON MICROSCOPYf_dihedral_angle_d3.9761218
ELECTRON MICROSCOPYf_chiral_restr0.0391498
ELECTRON MICROSCOPYf_plane_restr0.0031522

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