1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Functional and structural characterization of an ECF-type ABC transporter for vitamin B12

  1. Joana A Santos
  2. Stephan Rempel
  3. Sandra TM Mous
  4. Cristiane T Pereira
  5. Josy ter Beek
  6. Jan-Willem de Gier
  7. Albert Guskov  Is a corresponding author
  8. Dirk J Slotboom  Is a corresponding author
  1. University of Groningen, The Netherlands
  2. University of Campinas, South America
  3. Stockholm University, Sweden
Research Article
Cite this article as: eLife 2018;7:e35828 doi: 10.7554/eLife.35828
5 figures, 1 table, 1 data set and 3 additional files

Figures

Figure 1 with 1 supplement
Structures of cobalamin and cobinamide.

(a) Cobalamin structure, represented in the base-on conformation with the 5’,6’-dimethyl-benzimidazole ribonucleotide moiety (α-ligand) coordinating the central cobalt ion. The variable β-ligands are denoted as R in the lower left corner. (b) Structure of cobinamide, which lacks the DMBI moiety and has two cyano groups coordinating the cobalt ion from each side of the corrin ring.

https://doi.org/10.7554/eLife.35828.002
Figure 1—figure supplement 1
3D structures of cobalamin and cobinamide.

Cyanocobalamin (green) and mono-cyanocobinamide (cyan) are shown in stick representation. The central cobalt ion is colored in grey, nitrogen atoms in blue, oxygen atoms in red and phosphate atoms in orange.

https://doi.org/10.7554/eLife.35828.003
ECF-CbrT supports cobalamin-dependent growth of an E.coli deletion strain.

(a) The triple knock out strain E. coli ΔFEC expressing the BtuCDF ABC-transporter (positive control) grows in the presence of 50 μg/ml L-methionine or 1 nM CN-Cbl with lag-times of 300 min or 380 min, respectively, (b) E. coli ΔFEC carrying only the empty expression vector (negative control) grows only in the presence of 50 μg/ml L-methionine but not with 1 nM CN-Cbl. The lag-times of the negative controls are 450 min or >1000 min, respectively. (c) E. coli ΔFEC expressing the entire ECF-CbrT transporter supports growth in the presence of either 1 nM Cbl or 1 nM Cbi with a lag-time of 470 min or 730 min, respectively. The lag-time in the presence of 50 μg/ml L-methionine is 410 min. (d) Expression of the solitary S-component CbrT without its cognate ECF-module is not able to support growth of E. coli ΔFEC in the presence of 1 nM CN-Cbl.

https://doi.org/10.7554/eLife.35828.004
Figure 3 with 1 supplement
57Co-cyanocobalamin (cyano-Cbl) and 57Co-cobinamide (Cbi) transport by purified and reconstituted ECF-CbrT.

(a) ATP-dependent uptake of radiolabeled CN-Cbl and Cbi by ECF-CbrT in proteoliposomes. Proteoliposomes were loaded with either 5 mM Mg-ATP (circles for CN-Cbl, triangles for Cbi) or 5 mM Mg-ADP (CN-Cbl, squares). (b) Competition assay using Cbl-analogues. The initial uptake rate at 1 nM 57Co-cyanocobalamin (CN-Cbl) was measured. Competing compounds (adenosyl-cobalamin (Ado-Cbl); methyl-cobalamin (CH3-Cbl); hydroxyl-cobalamin (OH-Cbl); cobinamide or hemin) were added at a concentration of 250 nM. The uptake was normalized to a condition without competitor (10 pmol*mg−1*min−1). Since hemin is not readily soluble in an aqueous solution, we added 1% (v/v) DMSO during the assay, which did not affect the transporter activity. All competition experiments were performed in triplicate and the error bars indicate the standard deviation (s.d.).

https://doi.org/10.7554/eLife.35828.005
Figure 3—figure supplement 1
Kinetics of cobalamin uptake by ECF-CbrT.

Transport rates are shown as a function of the cobalamin concentration. Proteoliposomes were loaded with 5 mM Mg-ATP. Experiments were performed in triplicate and error bars indicate the s.d.’s. Values for the initial uptake rates were obtained by fitting the Michaelis-Menten function to the data, R2 = 0.979. The estimated KM value is 2.1 ± 0.4 nM and the Vmax is 0.06 ± 0.01 pmolmg−1s−1.

https://doi.org/10.7554/eLife.35828.006
Figure 4 with 1 supplement
Cobalamin and cobinamide binding to CbrT.

(a) Co-purification of CN-cobalamin with CbrT. The elution peak of the size exclusion column at a volume of 14 ml contains purified CbrT. The protein absorbs at 280 nm and CN-Cbl at 361 nm, showing that CbrT is eluted bound to CN-Cbl. (b) and (c) ITC measurements of Cbl and Cbi binding to CbrT. The determined Kd values for Cbl and Cbi were averaged from triplicate measurements and the error is s.d. (d) ITC measurement showing the absence of Cbl-binding to the full complex, ECF-CbrT. Fitting of single binding site models to the data is shown in panel (e).

https://doi.org/10.7554/eLife.35828.007
Figure 4—figure supplement 1
Binding of Cbl-analogs to CbrT.

ITC measurements of OH-Cbl (a) and CH3-Cbl (b) binding to CbrT. The determined Kd values were averaged from duplicate measurements. Fitting of single binding site models to the data is shown in panel c).

https://doi.org/10.7554/eLife.35828.008
Comparison of the structures of ECF-CbrT and ECF-FolT from L.delbrueckii.

(a) Cartoon representation of ECF–CbrT from the perspective of the plane of the membrane. Cytoplasmic ATPases, EcfA and EcfA’, are colored in red, EcfT in cyan and CbrT in yellow. (b) Structural differences between the membrane domains of EcfT. The structures of ECF-CbrT (cyan) and ECF-FolT2 (green) from L. delbrueckii were superimposed by structural alignment of the ATPase units. Pro71 of EcfT is represented in sticks. (c) and (d) Surface representation of CbrT (c, yellow) and FolT2 (d, orange) interacting with EcfT (cartoon representation coloured in grey) with loop 3 of the S-components colored in blue. A dashed line highlights the movement of transmembrane helix 3 of EcfT. (e) and (f) Loop 3 obstructs access to the substrate binding cavity in CbrT but not in FolT2. (e) Slice-through of CbrT in surface representation, viewed from the plane of the membrane. Loop 3 is colored in blue. The ECF module has been omitted for clarity. (f) Same slice through representation like in (e) but for FolT2.

https://doi.org/10.7554/eLife.35828.009

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene
(Lactobacillus delbrueckii
subsp. bulgaricus)
cbrTNALDB_RS00385
Strain, strain
background (E. coli)
MC1061Casadaban, M. J., and
Cohen, S. N. (1980).
Analysis of gene control
signals by DNAfusion
and cloning in Escherichia
coli.Journal of Molecular
Biology, 138(2),179–207
PMID 6997493
E. coli ΔFEC was constructed in
this paper with the following
deletions ΔbtuF, ΔmetE, and ΔbtuC::
KmR. Strain requires either L-methionine
or cobalmin/cobinamide plus expression
of an appropiate cobalmin/cobinamide
transporter. Strain can be made available
upon reasonable request.
Strain, strain
background (E. coli)
JW0154Coli Genetic Stock
Center Yale
E. coli ΔFEC was constructed in this
paper with the following deletions
ΔbtuF, ΔmetE, and ΔbtuC::KmR. Strain
requires either L-methionine or
cobalmin/cobinamide plus expression
of an appropiate cobalmin/cobinamide
transporter. Strain can be made available
upon reasonable request.
Strain, strain
background (E. coli)
JW3805Coli Genetic Stock
Center Yale
E. coli ΔFEC was constructed in this
paper with the following deletions ΔbtuF,
ΔmetE, and ΔbtuC::KmR. Strain requires
either L-methionine or cobalmin/cobinamide
plus expression of an appropiate cobalmin/
cobinamide transporter. Strain can be
made available upon reasonable request.
Strain, strain
background (E. coli)
JW1701Coli Genetic Stock
Center Yale
E. coli ΔFEC was constructed in this paper
with the following deletions ΔbtuF, ΔmetE,
and ΔbtuC::KmR. Strain requires either
L-methionine or cobalmin/cobinamide
plus expression of an appropiate cobalmin/
cobinamide transporter. Strain can be
made available upon reasonable request.
Strain, strain
background (E. coli)
ΔFECThis paperE. coli ΔFEC was constructed in this paper
with the following deletions ΔbtuF, ΔmetE,
and ΔbtuC::KmR. Strain requires either
L-methionine or cobalmin/cobinamide
plus expression of an appropiate cobalmin/
cobinamide transporter. Strain can be
made available upon reasonable request.
Biological sample
(Lactobacillus delbrueckii)
Lactobacillus delbrueckii
subsp. bulgaricus genomic DNA
DSMZDSM 20081
Recombinant
DNA reagent
pBAD24_CbrTThis paperExpression plasmids for CbrT and
ECF-CbrT in E. coli. Plasmids can be
provided upon reasonable request.
Recombinant
DNA reagent
p2BAD_ECF_CbrTThis paperExpression plasmids for CbrT and
ECF-CbrT in E. coli. Plasmids can be
provided upon reasonable request.
Chemical
compound, drug
CN-CblAcros405920050
Chemical
compound, drug
OH-CblSigma-Aldrich95200–1G
Chemical
compound, drug
Met-CblSigma-AldrichM9756-250G
Chemical
compound, drug
Ado-CblSigma-AldrichC0884-250MG
Chemical
compound, drug
CbiSigma-AldrichC3021-50MG
Chemical
compound, drug
heminSigma-Aldrich51280–1G
Chemical
compound, drug
57Co-cyanocobalaminMP-Biomedicals06B-430000
Chemical
compound, drug
perchloric acidSigma-Aldrich311421–50 ML
Software,
algorithm
Origin 8Company
OtherECF-CbrT coordinate file
and structure factors
this paperaccession numberPDB ID code 6FNPCrystal structure of ECF-CbrT

Data availability

Diffraction data have been deposited in PDB under the accession code 6FNP.

The following data sets were generated
  1. 1

Additional files

Supplementary file 1

Data collection, phasing and refinement statistics.

https://doi.org/10.7554/eLife.35828.010
Supplementary file 2

Primer list used in this study.

https://doi.org/10.7554/eLife.35828.011
Transparent reporting form
https://doi.org/10.7554/eLife.35828.012

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