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Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog

Abstract

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

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Fig. 1: Design for 19F and Ni2+ labeling of GltPh for R1 PRE.
Fig. 2: 19F-NMR spectra of dHis/M385C-TET GltPh and its mutants.
Fig. 3: 19F peak assignment using Ni2+-mediated PRE.
Fig. 4: Paramagnetic R1 relaxation and conformational exchange of the K290A mutant.
Fig. 5: Paramagnetic R1 relaxation and conformational exchange of the RSMR mutant.
Fig. 6: Intrinsic relaxation rate R1,A* determines the range of accessible exchange rates.

Data availability

Atomic coordinates for the cryo-EM structures of the OFS and iOFS states have been deposited in the Protein Data Bank under accession codes 6UWF and 6UWL, respectively, and the corresponding cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-20922 and EMD-20923, respectively. The other data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank X. Yao for helpful discussions, M. Goger and S. Bhattacharya for help with setting up NMR and M.A. Cuendet for help with setting up initial MD simulations. We thank C. Xu and K. Song at the UMass cryo-EM facility for help with electron microscopy data collection. We also thank W. Eng for assistance with protein expression. This work was supported by NIH grants R37NS085318 and R01NS064357 (O.B.), R37AG019391 (D.E.) and S10OD016320 (C.B.). O.B., D.E. and C.B. are members of the New York Structural Biology Center (NYSBC), which is supported in part by NIH grant P41 GM118302 (CoMD/NMR, Center on Macromolecular Dynamics by NMR Spectroscopy), ORIP/NIH facility improvement grant CO6RR015495 and NIH grant S10OD018509. The coordinates of the structures and the density maps have been submitted to the Protein Data Bank under the accession codes of 6UWF and 6UWL.

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Contributions

Y.H., D.E. and O.B. designed the experiments. Y.H. and G.L. performed the NMR experiments. Y.H. and O.B analyzed the data. C.B. assisted with NMR experimental design, data collection and analysis. X.W. performed cryo-EM imaging, analyzed data and refined molecular models. G.H.M.H. performed transport activity assays. A.M.R. performed molecular dynamics simulations. Y.H., X.W., A.M.R., O.B., D.E., H.W. and C.B. wrote the manuscript.

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Correspondence to David Eliezer or Olga Boudker.

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Extended data

Extended Data Fig. 1 Protein purity, and 3H L-asp uptake and simulated 19F-Ni2+ distance distribution.

a, Scheme for site-specifically introducing 19F label into M385C GltPh mutant. b, Representative size exclusion chromatography elution profile of M385C-TET GltPh. More than 3 independent samples were repeated with similar results. c, SDS-PAGE gel imaged by Coommasie blue staining (middle) and fluorescence (right) and of M385C GltPh labeled with fluorescein-5-malaimide before (lane 1) and after (lane 2) labeling with TFET. Protein samples were incubated with 10-fold excess of fluorescein-5-maleimide for 4 h prior to analysis. Two independent samples were prepared and yielded similar results. d. Michaelis-Menten kinetics of 3H L-Asp uptake for wide type GltPh (black circle), M385C-TET (red square), and dHis/M385C-TET GltPh (blue triangle). Data shown are means ± s.d. (N = 3 biological replicates). e, Distance probability distributions between 19F and Ni2+ calculated from 100 ns of the molecular dynamics simulation trajectories. To mimic experimental conditions M385 was mutated to NMR probe TET, residues 215 and 219 were mutated to histidine, and Zn2+ ion was constrained between these histidines (see Online Methods for details). The distance distributions were calculated for all three protomers and shown curves are averaged values from three protomers in the OFS (left) and the IFS (right). DTDP: 2,2’-dithiodipyridine; TFET: trifluoroethanethiol. Source data

Extended Data Fig. 2 Specific Ni2+ binding to dHis/M385C-TET GltPh mutant.

1D 19F-NMR spectra of M385C-TET GltPh (a) and dHis/M385C-TET GltPh (b) without (up) and with (bottom) 3 molar equivalents of Ni2+ ions. Spectra were recorded at 293 K in the presence of NaCL and L-asp. Raw data are black, fits are magenta and deconvoluted peaks are blue. Note: the dHis/M385C-TET spectra are the same as the ones shown in Fig. 2a and Fig. 3a in the main text.

Extended Data Fig. 3 Cryo-EM data processing.

a, Angular distribution of particles contributing to the final reconstitution. Number of views at each angular orientation is represented by length and color of cylinders where red indicates more views. b, Final maps after Relion post-processing colored according to local resolution estimation using ResMap. c, Fourier shell correlation (FSC) curves indicating the resolution at the 0.143 threshold of final masked (black) and unmasked (orange) maps of GltPh OFS (left) and iOFS (right). d, FSC curves from cross-validation of refined GltPh OFS (left) and iOFS (right) models compared to the masked half-map 1 (Orange traces: FSCwork, used during validation refinement), masked half-map 2 (Blue traces: FSCfree, not used during validation refinement), and the masked summed map (Black traces: FSCsum). e, Data processing flow chart for GltPh reconstituted into nanodisc in the presence of NaCl and L-asp.

Extended Data Fig. 4 2D 19F EXSY spectrum of dHis/M385C-TET GltPh.

Spectrum was recorded with mixing time of 0.4 s in the presence of 200 mM Na+ and 10 µM L-asp at 298 K.

Supplementary information

Supplementary Information

Supplementary Note, Fig. 1 and Tables 1–2.

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Source data

Source Data Extended Data Fig. 1

Unprocessed SDS-PAGE gels.

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Huang, Y., Wang, X., Lv, G. et al. Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog. Nat Chem Biol 16, 1006–1012 (2020). https://doi.org/10.1038/s41589-020-0561-6

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