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- PDB-8k7a: Cryo-EM structure of nucleotide-bound ComA E647Q mutant with Mg2+ -

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Basic information

Entry
Database: PDB / ID: 8k7a
TitleCryo-EM structure of nucleotide-bound ComA E647Q mutant with Mg2+
ComponentsTransport/processing ATP-binding protein ComA
KeywordsTRANSPORT PROTEIN / ABC transporter / 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 C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / 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 C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / 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
ADENOSINE-5'-TRIPHOSPHATE / Transport/processing ATP-binding protein ComA
Similarity search - Component
Biological speciesStreptococcus pneumoniae R6 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 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
DepositionJul 26, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 11, 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.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2May 8, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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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)161,9276
Polymers160,8642
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 80432.039 Da / Num. of mol.: 2 / Mutation: L643I, E647Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae R6 (bacteria) / Strain: R6 / Gene: comA / Production host: Escherichia coli (E. coli)
References: UniProt: P59653, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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 (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 software
IDNameVersionCategory
7UCSF Chimera1.17.1model fitting
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47217 / Symmetry type: POINT

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