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ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism

Nature Structural & Molecular Biology volume 22, pages 565–571 (2015)Cite this article

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Abstract

ECF transporters are a family of active transporters for vitamins. They are composed of four subunits: a membrane-embedded substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT) and two ATP-binding-cassette ATPases (EcfA and EcfA′). We have investigated the mechanism of the ECF transporter for riboflavin from the pathogen Listeria monocytogenes, LmECF–RibU. Using structural and biochemical approaches, we found that ATP binding to the EcfAA′ ATPases drives a conformational change that dissociates the S subunit from the EcfAA′T ECF module. Upon release from the ECF module, the RibU S subunit then binds the riboflavin transport substrate. We also find that S subunits for distinct substrates compete for the ATP-bound state of the ECF module. Our results explain how ECF transporters capture the transport substrate and reproduce the in vivo observations on S-subunit competition for which the family was named.

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Figure 1: TMRM labeling of LmRibU and LmECF–RibU.
Figure 2: Crystal structure of AMP-PNP–bound EcfAA′ heterodimer.
Figure 3: ATPase activity of LmECF–RibU.
Figure 4: Analytical SEC of WT and hydrolysis-deficient mutant LmECF–RibU.
Figure 5: ATP-dependent S-subunit exchange by LmECF.
Figure 6: The ATP-bound state of LmECF exchanges S subunits.
Figure 7: Proposed mechanism of group II ECF transporters.

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Acknowledgements

We are grateful to the staff at beamlines X25 and X29 of the National Synchrotron Light Source in the Brookhaven National Laboratory for assistance in X-ray diffraction experiments. We thank R. Mancusso, D. Sauer and C. Huang for technical assistance and J. Marden for comments on the manuscript. N.K.K. thanks the American Heart Association and the US National Institutes of Health (NIH) (F32-HL091618) for postdoctoral fellowships. This work was also financially supported by NIH grants R01DK099023, R01-DK073973, R01-GM093825 and R01- MH083840 to D.-N.W.

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  1. Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA

    Nathan K Karpowich, Jin Mei Song, Nicolette Cocco & Da-Neng Wang

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  1. Nathan K Karpowich
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N.K.K. designed and performed the experiments with J.M.S. and N.C.; N.K.K. and D.-N.W. analyzed the data and wrote the manuscript.

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Correspondence to Nathan K Karpowich or Da-Neng Wang.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the purified ECF riboflavin transporter from L. monocytogenes, LmECF–RibU.

(a) Preparative size exclusion chromatography (SEC) trace of purified LmECF–RibU and SDS-PAGE of the peak fractions (inset). (b) The molecular mass (Mw) of the protein complex in detergent was determined by analytical SEC and multi-angle laser light scattering (MALLS).

Supplementary Figure 2 TMRM labeling of WT and cysteine mutants of LmRibU.

(a) Purified LmRibU variants were incubated with the thiol reactive probe, TMRM, for the indicated time points. After termination of the reaction by DTT, samples were subjected to SDS-PAGE and TMRM labeling was visualized on a fluorescence scanner. (b) Initial rates of TMRM labeling were calculated from the data shown in (a).

Supplementary Figure 3 TMRM labeling of WT and cysteine mutants of LmECF–RibU.

(a) Ribbon diagram of the LmECF–RibU homology model with each subunit colored as indicated and cysteine residues shown as red spheres. The position of the membrane is represented as black lines. (b) Purified LmECF–RibU variants were labeled with the thiol reactive probe, TMRM, for the indicated time points. After SDS-PAGE (top panels), the labeling of the individual subunits was visualized on a fluorescence scanner (bottom panels). (c) Initial rates of TMRM labeling were calculated from these data.

Supplementary Figure 4 AMP-PNP is a low-affinity competitive inhibitor of LmECF–RibU.

(a) ATPase activity of LmECF–RibU in the presence of increasing concentrations of AMPPNP. Analytical SEC traces of LmECF–RibU measured in the presence of the vitamin B2 transport substrate and several concentrations of AMPPNP detected by UV absorbance (b) or riboflavin fluorescence (c).

Supplementary Figure 5 Characterization of the H199′A H204A ATPase-deficient mutant of LmECF–RibU.

(a) Preparative SEC trace of the purified protein and SDS-PAGE of the peak fractions (inset). (b) The molecular mass of the protein complex in detergent was determined by analytical SEC and MALLS.

Supplementary Figure 6 Schematic illustrating the experimental design to test for S-subunit exchange by EcfAA′T and calibration of the S-subunit exchange assay with free RibU*.

(a) First, a mixture of LmECF–RibU and isolated substrate-bound RibU that has been labeled with a fluorophore (S*) was prepared. Next either ADP, ATP, or no nucleotide is added and the samples are incubated for 10 min at 25 °C. Subsequently, the samples are separated by SEC, and binding of S* by EcfAA′T is detected by monitoring the S* fluorescence relative to elution position. SEC traces of several concentrations of RibU* detected by UV absorbance (b) and TMRM fluorescence(c). The integrated peak areas from (c) were used to calibrate the assay, as shown in (d).

Supplementary Figure 7 The TMRM label attached to RibU* is not transferred to LmECF–RibU during the incubation step of the exchange reaction.

LmECF–RibU (1), RibU* (2), or an equimolar mixture of the two in the absence (3) or presence of MgATP (4) were incubated under the same conditions of the exchange reaction. Instead of SEC, these samples were subject to SDS-PAGE (left), and the TMRM labeling was visualized on a fluorescence scanner (right). If the TMRM molecule was transferred from RibU* to LmECF–RibU, we would expect to see a fluorescent band in (4) corresponding to EcfA, which is labeled by TMRM on Cys11.

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Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Notes 1–3 (PDF 609 kb)

Supplementary Data Set 1

Complete gels for Figure 5c,f (PDF 2753 kb)

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Karpowich, N., Song, J., Cocco, N. et al. ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism. Nat Struct Mol Biol 22, 565–571 (2015). https://doi.org/10.1038/nsmb.3040

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