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.

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 4h prior to analysis. Two independent samples were prepared and yielded similar results. d. Michaelis-Menten kinetics of ³H L-Asp uptake for wide type (WT) 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.

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 293K 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: FSC_work, used during validation refinement), masked half map 2 (Blue traces: FSC_free, not used during validation refinement), and the masked summed map (Black traces: FSC_sum). 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 298K.

Figure 1:. Design for ¹⁹F and Ni²⁺ labeling of GltPh for R₁ PRE.

a, Cartoon representations of the structures of L-asp bound GltPh in the OFS (PDB accession code 2NWX) and IFS (accession code 3KBC). The structurally rigid scaffold domain is colored wheat and the dynamic transport domain is colored blue and red in the OFS and IFS, respectively. The substrate L-asp is shown as spheres. Pink and cyan circles represent the expected locations of the ¹⁹F label and bound Ni²⁺ ion, respectively. Only one of the three protomers was shown. b, Dependence of the longitudinal R₁ PRE on the distance between ¹⁹F and paramagnetic centers based on Equation 1, assuming τ_c is 213 ns, S² is 0.1 and τ_i is 20 ps. Other parameters are given in Supplementary Note. MTSL, (1-Oxyl-2,2,5,5-tetramethylpyrroline-3- methyl)methanethiosulfonate. Lines are colored red (Ni²⁺), blue (Cu²⁺), magenta (MTSL), black (Co²⁺).

Figure 2:. ¹⁹F-NMR spectra of dHis/M385C-TET GltPh and its mutants.

1D spectra of dHis/M385C-TET GltPh (WT) recorded at 293 K in the presence of 200 mM Na⁺ and 10 μM L-asp (a), 0.6 M Na⁺ only (b), 200 mM Na⁺ and 1 mM TBOA (c) or 200 mM Na⁺ and 110 μM TMA (d). 1D spectra of K290A/dHis/M385C-TET (e) and RSMR/dHis/M385C-TET (f) GltPh mutants in the presence of 200 mM Na⁺ and 10 μM L-asp. The spectra were deconvoluted into Lorentzian peaks S1, S2 and S3. Raw data are black, fits are magenta and deconvoluted peaks are blue.

Figure 3:. ¹⁹F peak assignment using Ni²⁺-mediated PRE.

a, 1D ¹⁹F spectra of Na⁺/L-asp-bound dHis/M385C-TET GltPh recorded at 293 K in the presence of 3 molar equivalents of Ni²⁺ ions. Raw data are black, fits are magenta and deconvoluted peaks are blue. Inset: Ni²⁺ titration of 69 μM protein. Data are plotted as peak S1 population. Solid red line is the fit to the quadratic binding equation. b, Representative R₁ relaxation traces for S1 (top), S2 (middle) and S3 peak (bottom) in the absence (black) and in the presence of Ni²⁺ ions (color). Solid lines through the data correspond to mono-exponential fits and the results of the fits are shown in Supplementary Table 1. Three independently prepared samples yielded similar results. c, R₁ rates of S1, S2 and S3 peaks of M385C-TET and dHis/M385C-TET GltPh constructs in the absence (yellow) and presence of Ni²⁺ ions and bound to L-asp (cyan) or TMA (gray). Individual data points are shown and the bars indicate means ± s.d. (N = 3). d, Structural comparison between Cryo-EM and crystal structures of GltPh in the OFS (left) and iOFS (right) conformations. Single protomers are viewed in the plain of the membrane and are depicted in cartoon representation. The crystal structures are colored gray, and the Cryo-EM structures are in color with scaffold domain wheat and the transport domain blue. Bound L-asp is shown as spheres and is colored by atom type. Two structurally well-defined lipid molecules are in stick representation, colored by atom type, and labeled Lipid 1 and 2. M385 is rendered as pink spheres. The location of the dHis motif is highlighted as cyan spheres. The orange line indicates the position of the HP2 tips.

Figure 4:. Paramagnetic R₁ relaxation and conformational exchange of K290A mutant.

a, 1D ¹⁹F spectra of K290A/dHis/M385C-TET GltPh in the absence (left) and presence (right) of Ni²⁺ ions. Raw data are black, fits are magenta and deconvoluted peaks are blue. b, Representative R₁ relaxation traces of S1 (top), S2 (middle) and S3 (bottom) in the absence of Ni²⁺ ions (black), or in the presence of Ni²⁺ and bound to L-asp (red for S1, blue for S2 and S3) or TMA (cyan). Solid lines represent mono-exponential fits with fitted R₁ values shown in Supplementary Table 1. Three independently prepared samples yielded similar results. c, ¹⁹F-¹⁹F EXSY spectra of K290A/dHis/M385C-TET GltPh bound to L-asp (left) or TMA (right). Mixing time was set to 0.4 s. Dashed lines indicate cross peaks for S1 and S2 (dark blue), S1 and S3 (light blue) and S2 and S3 (black). d, R₁ rates of S1, S2 and S3 peaks of K290A/dHis/M385C-TET in the absence (yellow) and presence of Ni²⁺ ions and bound to L-asp (cyan) or TMA (gray). Individual data points are shown and the bars indicate means ± s.d. (N = 3).

Figure 5:. Paramagnetic R₁ relaxation and conformational exchange of RSMR mutant.

a, 1D ¹⁹F NMR spectra of RSMR/dHis/M385C-TET in the absence (left) and presence (right) of Ni²⁺ ions. Raw data are black, fits are magenta and deconvoluted peaks are blue. The red, black and blue arrows indicate the saturation pulse, control pulse and observed peak, respectively, in the STD experiment in panel d. b, Representative R₁ relaxation traces of S1 (top), S2 (middle) and S3 (bottom) peaks in the absence (black) and presence (red for S1 and blue for S2 and S3) of Ni²⁺ ions. Solid lines represent mono-exponential fits with fitted R₁ values shown in Supplementary Table 1. All measurements are in the presence of 200 mM NaCl and 10 μM L-asp. Three independently prepared samples yielded similar results. c, ¹⁹F-¹⁹F EXSY spectrum of RSMR/dHis/M385C-TET GltPh in the presence of 200 mM NaCl and 10 μM L-asp recorded with mixing time of 0.4 s. d, Decay of the S3 peak upon saturating the S1 peak (red arrow in a) in the STD experiment (red squares). To account for the off-resonance saturation effect, a control experiment (black circles) was performed at an equidistant frequency to S3 peak (black arrow in a). The effective decay curve (blue triangle) is fit to the Equation 5, with results given in Supplementary Table 1. Two independently prepared samples yielded similar results. e, R₁ rates of S1, S2 and S3 peaks of RSMR/dHis/M385C-TET in the absence (yellow) and presence of Ni²⁺ ions with NaCl and L-asp (cyan). Individual data points are shown and the bars indicate means ± s.d. (N = 3).

Figure 6:. Intrinsic relaxation rate R_1,A* determines the range of accessible exchange rates.

a, Simulated R₁ relaxation curves of spin B (R_1,B* = 3.0 s⁻¹) in exchange with A with R_1,A* = 10 s⁻¹ (top) or 100 s⁻¹ (bottom). The fraction of spins in state B was set to 0.15 and the rate of transition from state B to state A, k_BA, varied from 0 to 200 s⁻¹ (black 0, red 1, blue 2, green 5, cyan 10, magenta 20, brown 50, orange 100, purple 200). b, 1D ¹⁹F NMR spectra of dHis/A381C-TET GltPh in the absence (top) and presence (bottom) of 3 molar equivalents of Ni²⁺ ions. Raw data are black, fits are magenta and deconvoluted peaks are blue. c, Paramagnetic R₁ relaxation curve of the S0 (black circles), S1 (red squares), S2 (blues triangles) and S3 (green reverse triangles) peaks of dHis/A381C-TET GltPh in the presence of Ni²⁺ ions. All measurements were performed in the presence of 200 mM NaCl and 10 μM L-asp. Solid lines represent bi-exponential fits for the S0 and S1 peaks and mono-exponential fits for the S2 and S3 peaks. The fitted parameters for S0 are: k_fast = 124.7 ± 32.0 s⁻¹, k_slow = 2.2 ± 1.7 s⁻¹; S1: k_fast = 97.5 ± 5.4 s⁻¹, k_slow = 2.8 ± 0.5 s⁻¹; S2: k = 5.4 ± 0.8 s⁻¹; S3: k = 4.5 ± 0.5 s⁻¹. The bi-exponential nature of R₁ relaxations of peaks S0 and S1 may reflect the presence of small but significant chemical exchange with the low-PRE peaks. Two independently prepared samples yielded similar results.