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- PDB-7qe5: Structure of the membrane domains of the sialic acid TRAP transpo... -

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Basic information

Entry
Database: PDB / ID: 7qe5
TitleStructure of the membrane domains of the sialic acid TRAP transporter HiSiaQM from Haemophilus influenzae
Components
  • Megabody3
  • Sialic acid TRAP transporter permease protein SiaT
KeywordsTRANSPORT PROTEIN / membrane transporter / TRAP / sialic acid / elevator
Function / homologyTRAP transporter large membrane protein DctM / TRAP transporter, small membrane protein DctQ / TRAP C4-dicarboxylate transport system permease DctM subunit / Tripartite ATP-independent periplasmic transporters, DctQ component / Tripartite ATP-independent periplasmic transporter, DctM component / transmembrane transporter activity / plasma membrane / Sialic acid TRAP transporter permease protein SiaT
Function and homology information
Biological speciesHaemophilus influenzae (bacteria)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsPeter, M.F. / Hagelueken, G.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)HA 6805/5-1 Germany
German Research Foundation (DFG)HA 6805/4-1 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter.
Authors: Martin F Peter / Jan A Ruland / Peer Depping / Niels Schneberger / Emmanuele Severi / Jonas Moecking / Karl Gatterdam / Sarah Tindall / Alexandre Durand / Veronika Heinz / Jan Peter ...Authors: Martin F Peter / Jan A Ruland / Peer Depping / Niels Schneberger / Emmanuele Severi / Jonas Moecking / Karl Gatterdam / Sarah Tindall / Alexandre Durand / Veronika Heinz / Jan Peter Siebrasse / Paul-Albert Koenig / Matthias Geyer / Christine Ziegler / Ulrich Kubitscheck / Gavin H Thomas / Gregor Hagelueken /
Abstract: Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two ...Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.
History
DepositionDec 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 27, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sialic acid TRAP transporter permease protein SiaT
B: Megabody3


Theoretical massNumber of molelcules
Total (without water)129,1702
Polymers129,1702
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1700 Å2
ΔGint-6 kcal/mol
Surface area35650 Å2

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Components

#1: Protein Sialic acid TRAP transporter permease protein SiaT / N-acetylneuraminic acid permease / N-acetylneuraminic acid transporter / Neu5Ac permease


Mass: 67636.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haemophilus influenzae (bacteria) / Strain: ATCC 51907 / DSM 11121 / KW20 / Rd / Gene: siaT, siaQM, HI_0147 / Production host: Escherichia coli (E. coli) / References: UniProt: P44543
#2: Antibody Megabody3


Mass: 61534.348 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)

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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: HiSiaQM/Megabody complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Haemophilus influenzae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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 defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50.213 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20_4444refinement
PHENIX1.20_4444refinement
EM software
IDNameVersionCategory
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 215956 / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00615864
ELECTRON MICROSCOPYf_angle_d1.28587967
ELECTRON MICROSCOPYf_chiral_restr0.0631943
ELECTRON MICROSCOPYf_plane_restr0.0113975
ELECTRON MICROSCOPYf_dihedral_angle_d8.7891787

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