Abstract
Energy-coupling factor (ECF) transporters are a large family of ATP-binding cassette transporters recently identified in microorganisms. Responsible for micronutrient uptake from the environment, ECF transporters are modular transporters composed of a membrane substrate-binding component EcfS and an ECF module consisting of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding/hydrolysis components EcfA/A'. ECF transporters are classified into groups I and II. Currently, the molecular understanding of group-I ECF transporters is very limited, partly due to a lack of transporter complex structural information. Here, we present structures and structure-based analyses of the group-I cobalt ECF transporter CbiMNQO, whose constituting subunits CbiM/CbiN, CbiQ, and CbiO correspond to the EcfS, EcfT, and EcfA components of group-II ECF transporters, respectively. Through reconstitution of different CbiMNQO subunits and determination of related ATPase and transporter activities, the substrate-binding subunit CbiM was found to stimulate CbiQO's basal ATPase activity. The structure of CbiMQO complex was determined in its inward-open conformation and that of CbiO in β, γ-methyleneadenosine 5′-triphosphate-bound closed conformation. Structure-based analyses revealed interactions between different components, substrate-gating function of the L1 loop of CbiM, and conformational changes of CbiO induced by ATP binding and product release within the CbiMNQO transporter complex. These findings enabled us to propose a working model of the CbiMNQO transporter, in which the transport process requires the rotation or toppling of both CbiQ and CbiM, and CbiN might function in coupling conformational changes between CbiQ and CbiM.
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Acknowledgements
We thank the staff members at BL19U/18U of National Center for Protein Science Shanghai (NCPSS) and BL17U of Shanghai Synchrotron Radiation Facility (SSRF) for technical assistance in data collection, and the staff at the core facility center of Institute of Plant Physiology and Ecology for X-ray diffraction experimental test and analysis. We thank Dr David Degen from Rutgers University for manuscript reading and revision. This work was supported by grants from the Ministry of Science and Technology of China (2015CB910900 to PZ and 2015CB910104 to JW), the National Natural Science Foundation of China (31670755 and 31322016 to PZ), the Chinese Academy of Sciences (QYZDB-SSW-SMC006 to PZ and 2015LH0027 to FH), and the Shanghai Municipal Science and Technology Commission. Research in DL Lab is supported by the 1000 Young Talent Program, the Shanghai “Pujiang Talent” Program (15PJ1409400), and the National Natural Science Foundation of China (31570748).
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Supplementary information
Supplementary information, Figure S1
SDS-PAGE and western blot results of the samples during the purification process. (PDF 275 kb)
Supplementary information, Figure S2
Representative 2FoFc electron density contoured at 1.5σ level. (PDF 307 kb)
Supplementary information, Figure S3
Residue Phe75 from TM4 docks its side chain into the empty substrate binding site of CbiM (PDF 121 kb)
Supplementary information, Figure S4
Sequence alignment of CbiQ, NikQ and group-II EcfT from different species. (PDF 464 kb)
Supplementary information, Figure S5
Hydrogen-bonding Interactions between CH2-CH3 of CbiQ and CbiO. (PDF 448 kb)
Supplementary information, Figure S6
Sequence alignment of CbiM and NikM. (PDF 477 kb)
Supplementary information, Figure S7
A model shows the uncoupling of ATPase activity with cobalt transport caused by loss of CbiN. (PDF 113 kb)
Supplementary information, Table S1
Statistics of data collection and structure refinement. (PDF 108 kb)
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Bao, Z., Qi, X., Hong, S. et al. Structure and mechanism of a group-I cobalt energy coupling factor transporter. Cell Res 27, 675–687 (2017). https://doi.org/10.1038/cr.2017.38
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DOI: https://doi.org/10.1038/cr.2017.38
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