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- PDB-8pnl: Outward-open conformation of a Major Facilitator Superfamily (MFS... -

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Basic information

Entry
Database: PDB / ID: 8pnl
TitleOutward-open conformation of a Major Facilitator Superfamily (MFS) transporter MHAS2168, a homologue of Rv1410 from M. tuberculosis, in complex with an alpaca nanobody
Components
  • Nb_H2
  • Putative triacylglyceride transporter
KeywordsTRANSPORT PROTEIN / Major Facilitator Superfamily transporter / Nanobody / Outward-open conformation / Triacylglyceride extraction
Function / homology
Function and homology information


transmembrane transporter activity / membrane
Similarity search - Function
Sugar transport proteins signature 1. / Sugar transporter, conserved site / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Putative triacylglyceride transporter
Similarity search - Component
Biological speciesMycolicibacterium hassiacum DSM 44199 (bacteria)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsRemm, S. / Schoeppe, J. / Hutter, C.A.J. / Gonda, I. / Seeger, M.A.
Funding support Switzerland, European Union, 3items
OrganizationGrant numberCountry
Swiss National Science FoundationPP00P3_144823 Switzerland
European Research Council (ERC)consolidator grant nr 772190European Union
University of Zurichgrant nr. FK-17-035 Switzerland
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for triacylglyceride extraction from mycobacterial inner membrane by MFS transporter Rv1410.
Authors: Sille Remm / Dario De Vecchis / Jendrik Schöppe / Cedric A J Hutter / Imre Gonda / Michael Hohl / Simon Newstead / Lars V Schäfer / Markus A Seeger /
Abstract: Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein ...Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 Å structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude ~20 Å into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The functional role of the periplasmic helix extensions is to channel the extracted TAG into the lipid binding pocket of LprG.
History
DepositionJun 30, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / reflns / reflns_shell
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 / _citation_author.identifier_ORCID / _citation_author.name / _reflns.pdbx_CC_half / _reflns_shell.Rmerge_I_obs / _reflns_shell.pdbx_CC_half / _reflns_shell.pdbx_Rrim_I_all

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative triacylglyceride transporter
B: Nb_H2
C: Putative triacylglyceride transporter
D: Nb_H2


Theoretical massNumber of molelcules
Total (without water)140,2334
Polymers140,2334
Non-polymers00
Water0
1
A: Putative triacylglyceride transporter
B: Nb_H2


Theoretical massNumber of molelcules
Total (without water)70,1172
Polymers70,1172
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2070 Å2
ΔGint-12 kcal/mol
Surface area25240 Å2
2
C: Putative triacylglyceride transporter
D: Nb_H2


Theoretical massNumber of molelcules
Total (without water)70,1172
Polymers70,1172
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1980 Å2
ΔGint-14 kcal/mol
Surface area26080 Å2
Unit cell
Length a, b, c (Å)57.780, 160.700, 82.960
Angle α, β, γ (deg.)90.00, 109.01, 90.00
Int Tables number4
Space group name H-MP1211
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Putative triacylglyceride transporter / Sugar (And other) transporter family protein


Mass: 57082.887 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium hassiacum DSM 44199 (bacteria)
Gene: C731_2106, MHAS_02168 / Plasmid: pACE_C3GH_MHAS2168
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: K5B8L6
#2: Antibody Nb_H2


Mass: 13033.628 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Nanobody / Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pBXNPHM3_Nb_H2 / Production host: Escherichia coli MC1061 (bacteria)

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

Crystal
IDDensity Matthews (Å3/Da)Density % sol (%)
12.652.63
2
Crystal grow
Temperature (K)Crystal-IDMethodpHDetails
293.151lipidic cubic phase5.7380 mM NaH2PO4, 0.1 M sodium citrate (pH 5.7), 28% (v/v) PEG400, 2.4% (v/v) 1,4-butanediole
293.152lipidic cubic phase5.8420 mM NaH2PO4, 0.1 M sodium citrate (pH 5.8), 28% (v/v) PEG400, 2.4% (v/v) 1,4-butanediole

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 0.999 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 24, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.999 Å / Relative weight: 1
ReflectionResolution: 2.7→44.7 Å / Num. obs: 39032 / % possible obs: 99.3 % / Redundancy: 4.65 % / CC1/2: 0.999 / Rmerge(I) obs: 0.095 / Rrim(I) all: 0.107 / Net I/σ(I): 8.77
Reflection shell
Resolution (Å)Rmerge(I) obsNum. unique obsCC1/2Rrim(I) allDiffraction-ID
2.7-2.771.63829110.3471.8381
2.77-2.851.34528020.4161.5091
2.85-2.930.99627310.5611.121
2.93-3.020.84826930.6560.9541
3.02-3.120.6625750.7630.7421
3.12-3.230.5224960.8220.5861
3.23-3.350.36324110.9030.4091
3.35-3.490.28223180.9370.3191
3.49-3.640.21122130.9560.241
3.64-3.820.15420890.9780.1761
3.82-4.020.11820130.9890.1321
4.02-4.270.09118980.9940.1021
4.27-4.560.07617840.9940.0861
4.56-4.930.06616840.9960.0741
4.93-5.40.06715260.9970.0761
5.4-6.040.07214050.9950.0821
6.04-6.970.05512110.9970.0631
6.97-8.540.03610210.9990.0411
8.54-12.070.0278110.9990.0311
12.07-500.0264400.9990.0291

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.7→44.7 Å / SU ML: 0.39 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 32.66 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2915 1952 5 %
Rwork0.245 --
obs0.2474 39014 99.42 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.7→44.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8824 0 0 0 8824
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003
X-RAY DIFFRACTIONf_angle_d0.729
X-RAY DIFFRACTIONf_dihedral_angle_d12.6153111
X-RAY DIFFRACTIONf_chiral_restr0.0421481
X-RAY DIFFRACTIONf_plane_restr0.0091536
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7-2.770.39141410.35522673X-RAY DIFFRACTION100
2.77-2.840.32621380.31552620X-RAY DIFFRACTION100
2.84-2.930.3171390.29122655X-RAY DIFFRACTION100
2.93-3.020.37051410.29492679X-RAY DIFFRACTION100
3.02-3.130.33321390.29082633X-RAY DIFFRACTION100
3.13-3.250.3021400.27182654X-RAY DIFFRACTION100
3.25-3.40.30441400.25082662X-RAY DIFFRACTION100
3.4-3.580.28081380.24532636X-RAY DIFFRACTION100
3.58-3.80.2941380.25612611X-RAY DIFFRACTION98
3.81-4.10.33951400.24872663X-RAY DIFFRACTION100
4.1-4.510.31161380.23082632X-RAY DIFFRACTION99
4.51-5.160.29331400.23642658X-RAY DIFFRACTION99
5.16-6.50.30581400.26882643X-RAY DIFFRACTION99
6.5-44.70.22141400.20272643X-RAY DIFFRACTION98

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