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Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter

Fan, Chengcheng and Kaiser, Jens T. and Rees, Douglas C. (2019) Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter. . (Unpublished)

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ATP Binding Cassette (ABC) transporters often exhibit significant basal ATPase activity in the absence of transported substrates. To investigate the factors that contribute to this inefficient coupling of ATP hydrolysis to transport, we characterized the structures and functions of variants of the bacterial Atm1 homolog from Novosphingobium aromaticivorans (NaAtm1), including forms with disulfide crosslinks between the nucleotide binding domains. Unexpectedly, disulfide crosslinked variants of NaAtm1 reconstituted into proteoliposomes not only transported oxidized glutathione, but also exhibited more efficient coupling of ATP hydrolysis to GSSG transport than the native transporter. These observations suggest that enhanced conformational dynamics of reconstituted NaAtm1 may contribute to the inefficient use of ATP. Understanding the origins of this uncoupled ATPase activity, and reducing the impact through disulfide crosslinking or other protocols, will be critical for the detailed dissection of ABC transporter mechanism to assure that the ATP dependent steps are indeed relevant to substrate translocation.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper
Fan, Chengcheng0000-0003-4213-5758
Kaiser, Jens T.0000-0002-5948-5212
Rees, Douglas C.0000-0003-4073-1185
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. bioRxiv preprint first posted online Nov. 9, 2019. Author contributions: C.F. and D.C.R designed the research; C.F. performed the research; C.F., J.T.K and D.C.R. analyzed the data; and C.F and D.C.R prepared the manuscript. The authors declare no competing interests. Data availability: Atomic coordinates were deposited in the Protein Data Bank with accession codes 6PAM (NaA527C-MgADP), 6PAN (NaS526C-ATP), 6PAO (NaT525C-ATP), 6PAQ (NaE523Q-ATP) and 6PAR (NaAtm1-MgAMPPNP). The raw data for ATPase and transport assays that support the findings in Figure 2 and Table 2 are included in Table S6, and all the other data are available from the corresponding author upon reasonable request. We thank the beamline staffs of the Stanford Synchrotron Radiation Lightsource beamline 12-2 and of the Advanced Photon Source GM/CA beamline for support during data collection. Discussions with Paul Adams, Gabriele Meloni, William Clemons, the organizers and speakers at the Cold Spring Harbor X-ray Method in Structural Biology Course (2018), the CCP4/APS School for Macromolecular Crystallography (2017) and the SBGrid/NE-CAT Phenix Workshop (2016) are gratefully acknowledged. We thank the Gordon and Betty Moore Foundation and the Beckman Institute for their generous support of the Molecular Observatory at Caltech. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The Eiger 16M detector was funded by an NIH–Office of Research Infrastructure Programs, High-End Instrumentation Grant (1S10OD012289-01A1).
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Department of Energy (DOE)DE-AC02-76SF00515
Department of Energy (DOE)DE-AC02-06CH11357
National Cancer InstituteUNSPECIFIED
National Institute of General Medical SciencesUNSPECIFIED
Record Number:CaltechAUTHORS:20191111-072609203
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Official Citation:Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter. Chengcheng Fan, Jens T. Kaiser, Douglas C Rees. bioRxiv 836676; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99761
Deposited By: Tony Diaz
Deposited On:12 Nov 2019 21:59
Last Modified:16 Nov 2021 17:49

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