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Published September 2015 | Accepted Version + Published
Journal Article Open

The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex


Despite the ubiquitous role of ATP-binding cassette (ABC) importers in nutrient uptake, only the Escherichia coli maltose and vitamin B_(12) ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250-253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATPbound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nm. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ~40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system.

Additional Information

© 2015 De Gruyter. Received February 18, 2015; accepted March 19, 2015; previously published online March 23, 2015. A Vietnam International Education Development scholarship from the Vietnam Ministry of Education and Training scholarship to P.T.N, an NSF Graduate Fellowship to Q.W.L., and support of NIH Predoctoral Training Grant T32 GM07737 to N.S.K., are gratefully acknowledged. We thank Allen Lee for generating the original MetNIQ constructs, Christoph Müller for MetNIQ discussions, and Dr. Jens Kaiser and Dr. Nathan Dalleska for assistance with crystallography and ICP-MS, respectively. We gratefully acknowledge the Gordon and Betty Moore Foundation and the Beckman Institute for their generous support of the Molecular Observatory at Caltech and the staff at Beamline 12–2, Stanford Synchrotron Radiation Lightsource (SSRL), for their assistance with data collection. Use of the SSRL, 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). This project benefited from the use of instrumentation made available by the Caltech Environmental Analysis Center. This work was supported in part by NIH grant GM045162.

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Published - Nguyen_2015p1127.pdf

Accepted Version - nihms729241.pdf


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