Visualizing multistep elevator-like transitions of a nucleoside transporter

Abstract

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

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Figure 1: The outward-facing conformation of CNTNWN149S,F366A.
Figure 2: The intermediate conformations of CNTNWN149L.
Figure 3: Cysteine cross-linking in membranes.
Figure 4: State-dependent changes in the transport domain.
Figure 5: Intracellular and extracellular gates.
Figure 6: Elevator mechanism of CNT.

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Acknowledgements

Data were collected at beamlines 24-ID-C and 22-ID in the Advanced Photon Source. We thank B. Kloss and W. Hendrickson from the Center on Membrane Protein Production and Analysis (COMPPÅ) for additional homologue screening, and F. Valiyaveetil, J. Richardson, and D. Richardson for manuscript reading. This work was supported by NIH R01 GM100984 (S.-Y.L.) and NIH R35 NS097241 (S.-Y.L.). Beamline 24-ID-C is funded by P41GM103403 and S10 RR029205. COMPPÅ is funded by P41 GM116799 (W. Hendrickson).

Author information

M.H. and Z.L.J. crystallized the protein and performed ITC and flux experiments. M.H. solved the structures and carried out cross-linking experiments. S.-Y.L. and M.H. wrote the paper. All authors discussed the results and commented on the manuscript.

Correspondence to Seok-Yong Lee.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of the human CNT isoforms with vcCNT and CNTNW.

Bars representing helices are coloured as in Fig. 1. Grey highlight indicates sequence conservation. Blue and green highlights indicate regions involved in state-dependent interactions between the scaffold and transport domains, respectively. The N-terminal 150–180 residues of the hCNTs are omitted for clarity as they are not present in vcCNT or CNTNW.

Extended Data Figure 2 Functional characterization of CNTNW and its structure in complex with sodium and uridine.

a, Radioactive uridine flux in proteoliposomes requires a sodium gradient. Average of n = 3 for wild-type and empty vesicles and n = 9 for mutants (technical replicates), error bar indicates s.e. b, Representative isothermal titration calorimetry raw data (top) and binding isotherm (bottom) for CNTNW (dissociation constant (Kd) = 4.5 μM, enthalpy change (ΔH°) = −8.1 kcal mol−1), CNTNWN149L (Kd = 847.5 μM, ΔH° = −1.8 kcal mol−1), CNTNWN149S (Kd = 30.8 μM, ΔH° = −2.4 kcal mol−1), and CNTNWN149S,F366A (no binding observed). Experiments were performed in triplicate (technical replicates). c, CNTNW trimer viewed from the intracellular side (left) and within the membrane plane (right). d, CNTNW viewed from the trimerization axis, coloured as in Fig. 1, with uridine in yellow and sodium in green. e, CNT topology diagram, coloured as in Fig. 1. f, g, Detailed view of the nucleoside-binding sites for CNTNW (f) and vcCNT (g). The configuration of the binding sites is nearly identical, except for Glu156 which adopts a different rotamer conformation in CNTNW. Source data

Extended Data Figure 3 Quality of the structure and electron density of the outward-facing structure.

a, 2FoFc simulated annealing (SA) composite omit map, calculated using 3,000 K, is shown at 1σ for the outward-facing protomer region in the CNTNWN149S,F366A crystal structure. The model is shown in ribbon representation with side chains in line representation where supported by the density, coloured as in Fig. 1. b, Cutout surface and cartoon representation of the outward-facing conformation, coloured as in Fig. 1. Two separate paths, outlined in yellow, provide access to either the sodium- or nucleoside-binding pockets. c, Cartoon representation of the crystal structures of two sodium- and nucleoside-binding mutants, CNTNWN149S,F366A (blue) and CNTNWN149S,E332A (green). The structures overlay with an overall Cα r.m.s.d. of 0.5 Å. CNTNW trimer viewed from within the membrane plane. The asterisk denotes the outward-facing protomer. d, Comparison of the outward-facing crystal structure (blue) with the repeat-swap-modelled outward-facing conformation (red, Protein Model DataBase PM0080188). In the crystal structure Phe366 (blue circle) was found to be 9 Å closer to the extracellular side of the membrane than in the modelled structure (red circle). The Cα r.m.s.d. of the transport domain alone is 7.9 Å, showing a substantial difference between the two structures.

Extended Data Figure 4 Experimentally phased map of CNTNWN149L-3 and anomalous signals from SeMet-labelled CNTNWN149L-1 and CNTNWN149L-3 guided model building.

a, b, An overview (a) and detailed view (b) of the electron density map of CNTNWN149L-3, solved by single anomalous dispersion phasing followed by solvent flattening at 3.55 Å. The experimentally phased map is shown in blue mesh at 1σ and the model is shown in ribbon representation with side chains as sticks where supported by the density, coloured as in Fig. 1. The asterisk denotes the outward-facing protomer. c, d, Anomalous difference Fourier maps were calculated using the MR phases of protomers A and B of CNTNWN149L-1 at 6 Å (c) and for CNTNWN149L-3 at 3.6 Å (d) (blue mesh at 3.5σ). Two mutants were designed to carry an additional methionine in HP1, CNTNWN149L,L159M (red mesh at 2.5σ in the intermediate 1 state at 4.6 Å) and HP2, CNTNWN149L,V328M (green mesh at 2.5σ in the intermediate 1 state at 5 Å and in the intermediate 3 state at 6 Å). The locations of methionine residues in the models of CNTNWN149L-1 and CNTNWN149L-3 agree well with the locations of Se anomalous peaks. e, The anomalous maps and models for intermediates 1 (beige) and 3 (blue) were overlaid to compare the locations of anomalous peaks. The positions of anomalous markers in the transport domain are in distinct locations in each intermediate structure. f, g, Close-up view of two methionine residues, Met168 and Met208, in the transport domains of intermediates 1 (beige) and 3 (blue) and their corresponding Se anomalous difference peaks.

Extended Data Figure 5 Quality of the electron density in the three CNTNWN149L crystal structures.

ac, A detailed view of the 2FoFc SA composite omit maps of the intermediate state protomers using 3,000 K at 1σ. df, FoFc SA omit maps, calculated at 3,000 K, shown at 2.2σ for HP1 (red), TM4 (orange), HP2 (blue), and TM7 (teal). Models are shown in ribbon representation with side chains as sticks where supported by the density, coloured as in Fig. 1. g, h, Each model fits poorly into the electron density of another intermediate state (2FoFc maps at 1). g, The models of CNTNWN149L-1 (green) and CNTNWN149L-2 (red) shown in the density of CNTNWN149L-2. CNTNWN149L-1 does not fit well in the density of CNTNWN149L-2. h, The models of CNTNWN149L-2 (red) and CNTNWN149L-3 (blue) shown in the density of CNTNWN149L-3. CNTNWN149L-2 does not fit well in the density of CNTNWN149L-3. i, Log-likelihood-gain (LLG) scores of molecular replacement, using MR-phaser, with the refined structure of each intermediate model in each of the data sets. The resolution was cut off at 4.1 Å to enable comparison of the LLG scores. For each intermediate the appropriate model has an LLG score substantially higher than the incorrect models.

Extended Data Figure 6 Crystal packing in the wild-type (inward-facing), CNTNWN149L-3 (intermediate), and CNTNWN149S,F366A (outward-facing) crystals.

Crystal contacts are mostly mediated by protomers A (red) and B (green) in each crystal. This provides protomer C (blue) with sufficient space to enable movement of the transport domain within the same crystal packing. Crystal packing interactions are shown as yellow surfaces.

Extended Data Figure 7 CNTNW cysteine cross-linking mutants, except for the negative control (CNTNWS109C,A373C), spontaneously cross-link to form covalent trimers.

a, Western blot of MBP-CNTNW cross-linking mutants in crude membrane preparations. Each cross-linking pair, except for the negative control, cross-links spontaneously to form covalently linked dimers and trimers, and this reaction is reversible by addition of reducing reagent. Western blots were replicated three times in the laboratory. b, Cross-linked CNTNW mutants have a similar elution volume in size exclusion chromatography as Cys-less CNT, as shown by size-exclusion chromatography and SDS–PAGE analysis. c, The purified cysteine cross-linking mutants were analysed by Coomassie-stained SDS–PAGE before and after PreScission protease treatment to remove the MBP-His tag. After PPX treatment, monomeric, dimeric, and trimeric CNTNW can be seen in the SDS–PAGE gel, indicated by the blue, green, and purple arrows, respectively. Air-oxidized protein in the peak fraction (asterisk) was reconstituted into proteoliposomes for the flux assay shown in Fig. 3.

Extended Data Figure 8 State-dependent interactions between the transport and scaffold domains.

a, Interactions between TM6 (grey) of the scaffold domain and transport domain elements HP2b (blue) and TM7b (teal) in the inward-facing, intermediate, and outward-facing conformations, coloured as in Fig. 1. b, Interactions between TM3 (grey) and transport domain elements TM4b (orange) and HP1b (red), coloured as in Fig. 1. The black box indicates the region shown in c. c, On the basis of its Cα location, Glu156, the only charged residue on the transport domain at this interface, appears to be positioned towards the interface with the scaffold domain in the intermediate states. The side chain is modelled as its ideal rotamer and a 2FoFc SA composite omit electron density map (blue mesh, 0.8σ) is shown. d, Cutout surface depictions show the changes in the specific interactions between TM6 and the transport domain elements HP2b and TM7b during the elevator motion. The interaction network is mostly made up of hydrophobic interactions.

Extended Data Figure 9 HP1 conformational transition and quality of the electron density.

a, Overlay of the transport domain for eight protomers (from the CNTNW, CNTNWN149L-1–3, and CNTNWN149S,F366A crystal structures) showing the transition between the inward-occluded, substrate-bound HP1 conformation (red trace) and the inward-facing-open HP1 conformation (purple trace). b, c, SA composite omit map (2FoFc maps at 1σ) around HP1 in the pre-translocation conformation in protomer A of CNTNWN149L-3 (b) and around HP1 in the inward-open conformation in protomer A of CNTNWN149L-1 (c). The electron density is shown as blue mesh. The ribbon representation is coloured as in Fig. 1, with side chains shown as lines where supported by the density.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Figure

This file contains the gel source data for Figure 3 and Extended Data Figure 7.

Multistep elevator-like transitions of a Concentrative Nucleoside Transporter from Neisseria wadsworthii

The transition from the inward-facing occluded conformation, through three intermediate states, to the outward-facing open conformation.

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Hirschi, M., Johnson, Z. & Lee, S. Visualizing multistep elevator-like transitions of a nucleoside transporter. Nature 545, 66–70 (2017) doi:10.1038/nature22057

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