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- PDB-8ttg: NorA single mutant - E222Q at pH 7.5 -

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Basic information

Entry
Database: PDB / ID: 8ttg
TitleNorA single mutant - E222Q at pH 7.5
Components
  • FabDA1 CDRH3 loop
  • Quinolone resistance protein NorA
KeywordsTRANSPORT PROTEIN / E222Q NorA / efflux pump / MRSA
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / plasma membrane
Similarity search - Function
Tetracycline resistance protein TetA/multidrug resistance protein MdtG / : / Multidrug resistance protein / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Quinolone resistance protein NorA
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsLi, J.P. / Li, Y. / Koide, A. / Kuang, H.H. / Torres, V.J. / Koide, S. / Wang, D.N. / Traaseth, N.J.
Funding support United States, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165782 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI108889 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS108151 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK135088 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/Office of the DirectorOD019994 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)RR029300 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121994 United States
CitationJournal: Nat Commun / Year: 2024
Title: Proton-coupled transport mechanism of the efflux pump NorA.
Authors: Jianping Li / Yan Li / Akiko Koide / Huihui Kuang / Victor J Torres / Shohei Koide / Da-Neng Wang / Nathaniel J Traaseth /
Abstract: Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to ...Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.
History
DepositionAug 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
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Revision 1.1Jul 3, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Quinolone resistance protein NorA
C: FabDA1 CDRH3 loop


Theoretical massNumber of molelcules
Total (without water)48,7282
Polymers48,7282
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Quinolone resistance protein NorA


Mass: 46654.863 Da / Num. of mol.: 1 / Mutation: E222Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: norA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q53459
#2: Protein/peptide FabDA1 CDRH3 loop


Mass: 2073.264 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
Has protein modificationN

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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: E222Q-NorA:FabDA1 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.100656 MDa / Experimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
2100 mMsodilum chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 52.96 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
4cryoSPARCv.3.3.1CTF correction
8PHENIXmodel refinement
10cryoSPARCv.3.3.1initial Euler assignment
11cryoSPARCv.3.3.1final Euler assignment
13cryoSPARCv.3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 246742 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023109
ELECTRON MICROSCOPYf_angle_d0.6074215
ELECTRON MICROSCOPYf_dihedral_angle_d3.816410
ELECTRON MICROSCOPYf_chiral_restr0.04484
ELECTRON MICROSCOPYf_plane_restr0.005513

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