Figures and data in Functional and structural characterization of an ECF-type ABC transporter for vitamin B12

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5 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement

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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

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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

Figure 2

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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

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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

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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 K_M value is 2.1 ± 0.4 nM and the V_max is 0.06 ± 0.01 pmolmg⁻¹s⁻¹.

https://doi.org/10.7554/eLife.35828.006

Figure 4 with 1 supplement

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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 *K_d* 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

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Binding of Cbl-analogs to CbrT.

ITC measurements of OH-Cbl (a) and CH₃-Cbl (b) binding to CbrT. The determined K_d 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

Figure 5

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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	Designation	Source or reference	Identifiers	Additional information
Gene (Lactobacillus delbrueckii subsp. bulgaricus)	cbrT	NA	LDB_RS00385
Strain, strain background (E. coli)	MC1061	Casadaban, 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)	JW0154	Coli 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)	JW3805	Coli 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)	JW1701	Coli 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)	ΔFEC	This paper		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.
Biological sample (Lactobacillus delbrueckii)	Lactobacillus delbrueckii subsp. bulgaricus genomic DNA	DSMZ	DSM 20081
Recombinant DNA reagent	pBAD24_CbrT	This paper		Expression plasmids for CbrT and ECF-CbrT in E. coli. Plasmids can be provided upon reasonable request.
Recombinant DNA reagent	p2BAD_ECF_CbrT	This paper		Expression plasmids for CbrT and ECF-CbrT in E. coli. Plasmids can be provided upon reasonable request.
Chemical compound, drug	CN-Cbl	Acros	405920050
Chemical compound, drug	OH-Cbl	Sigma-Aldrich	95200–1G
Chemical compound, drug	Met-Cbl	Sigma-Aldrich	M9756-250G
Chemical compound, drug	Ado-Cbl	Sigma-Aldrich	C0884-250MG
Chemical compound, drug	Cbi	Sigma-Aldrich	C3021-50MG
Chemical compound, drug	hemin	Sigma-Aldrich	51280–1G
Chemical compound, drug	57Co-cyanocobalamin	MP-Biomedicals	06B-430000
Chemical compound, drug	perchloric acid	Sigma-Aldrich	311421–50 ML
Software, algorithm	Origin 8	Company
Other	ECF-CbrT coordinate file and structure factors	this paper	accession numberPDB ID code 6FNP	Crystal structure of ECF-CbrT