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Evolutionary balance between foldability and functionality of a glucose transporter

Nature Chemical Biology volume 18, pages 713–723 (2022)Cite this article

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Abstract

Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3’s microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

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Fig. 1: Single-molecule magnetic tweezers assay for observing folding of single GLUT3 proteins.
Fig. 2: Identification of the folding order of GLUT3 domains.
Fig. 3: Dissection of the folding order of individual MFS folds.
Fig. 4: EMC facilitates folding of single GLUT3 proteins.
Fig. 5: PE lipids facilitate GLUT3 inter-domain assembly.
Fig. 6: Phylogenetic analysis reveals an asymmetric folding propensity across the N- and C-domains common for metazoan sugar transporters.

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Data availability

All data that support the findings of this study are available in the manuscript or supplementary figures. Raw data have been deposited in Github (https://github.com/tyyoonlab/ Nat_Chem_biol_NCHEMB - A210813554). The following PDB IDs were used (4ZWB, 4ZWC, 4ZW9, 4GBZ, 6RW3, 4LDS and 6H7D). The following UniProt IDs were used (P11169, P0AGF4, O97467, A0A0H2VG78 and Q9LT15). Source data are provided with this paper.

Code availability

A program, written in LabView, to control the magnetic tweezers apparatus has been deposited in Github (https://github.com/tyyoonlab/Science_aaw8208). Codes for analyzing the magnetic tweezers and FACS data has been deposited in Github (https://github.com/tyyoonlab/Nat_Chem_biol_NCHEMB-A210813554). Codes for estimating the helix insertion energy are available at Github under GPL3.0 license (https://github.com/schnamo/TMH_insertion_energy).

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Acknowledgements

We thank E. Kweon for help with preparing illustrations. This work was supported by National Creative Research Initiative Program (NRF-2021R1A3B1071354 to T.-Y.Y.), the Bio Medical Technology Development Program (NRF-2018M3A9E2023523 to T.-Y.Y.) and NRF grants (NRF-2019M3E5D6063903 and NRF-2020R1A2C2003783 to H.-J.C.; NRF-2019R1A6A1A10073437 and NRF-2020M3A9G7103933 to M.S.; NRF-2021M3A9I4021220 and NRF-2020R1A6C101A183 to S.-H.R), all funded by the National Research Foundation of South Korea. This work was also supported by the UK Medical Research Council (MRC_UP_12-1/10 to E.A.M.), the US National Science Foundation (MCB-181069 to W.I.) and National Institutes of Health grant (R01GM118684 to H.H.).

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Author notes

  1. These authors contributed equally: Hyun-Kyu Choi, Hyunook Kang, Chanwoo Lee.

Authors and Affiliations

  1. School of Biological Sciences, Seoul National University, Seoul, South Korea

    Hyun-Kyu Choi, Hyunook Kang, Chanwoo Lee, Hyun Gyu Kim, Charlotte Tumescheit, Sang Ah Kim, Hansol Lee, Soung-Hun Roh, Martin Steinegger, Hee-Jung Choi & Tae-Young Yoon

  2. Institute for Molecular Biology and Genetics, Seoul National University, Seoul, South Korea

    Hyun-Kyu Choi, Chanwoo Lee, Hyun Gyu Kim, Sang Ah Kim, Hansol Lee, Soung-Hun Roh & Tae-Young Yoon

  3. Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK

    Ben P. Phillips & Elizabeth A. Miller

  4. Departments of Biological Sciences and Chemistry, Lehigh University, Bethlehem, PA, USA

    Soohyung Park & Wonpil Im

  5. Department of Chemistry and Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI, USA

    Heedeok Hong

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Contributions

H.-K.C. and T.-Y.Y. conceived the project. H.-K.C., H.-J.C., E.A.M. and T.-Y.Y. designed the experiments. H.-K.C., C.L. and S.A.K. performed the magnetic tweezers experiment. H.K. and H.-J.C. expressed and purified GLUT3 proteins. B.P.P. and E.A.M. expressed and purified EMC. H.G.K. performed the flow cytometry experiment. S.P. and W.I. performed the molecular dynamics simulation. H.G.K., C.T. and M.S. performed the bioinformatic analysis. H.L. and S.-H.R. performed the TEM imaging. H.-K.C., C.L. and T.-Y.Y. prepared the manuscript, with assistance from H.-J.C., E.A.M. and H.H., and with input from all authors.

Corresponding authors

Correspondence to Elizabeth A. Miller, Hee-Jung Choi or Tae-Young Yoon.

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Extended data

Extended Data Fig. 1 Sample preparation of WT and C-domain-knotted GLUT3 proteins and bicelle membranes.

a, Elution profile obtained by size exclusion chromatography (SEC) of WT GLUT3 (black) and S265C/A469C GLUT3 (red). b, Purified WT GLUT3 protein analyzed by SDS-PAGE. The major peak position in (a) was used for the gels. The right lane is molecular weight standards. c, Representative gel image of SDS-PAGE after SYBR green staining. The left lane shows SpyCatcher-DNA handle only, while the right lane exhibits a mixture of the SpyCatcher-DNA handle and the purified Spytag-GLUT3-spytag. d, Bicelle size as measured by dynamic light scattering (DLS) under indicated lipid compositions. The data with gray color is a control sample of 1-µm polystyrene beads. e, Mean diameter of the bicelles determined for each lipid composition. Error bars represent SEM (n = 22 and 25 for 30 mol% PG and 100 mol% PG, respectively). f,g, Electron microscope images of bicelles with different lipid compositions. Two specified electron microscopy methods are used. h, Representative trace for the force-jump experiment to determine the refolding probability. Force was first increased to 25pN to induce full unraveling of the protein and then relaxed to 1 pN for 500 s before checking the folding status through re-application of 25 pN. The folding status was determined by the unfolding steps observed under 25pN. i, Probability for observing the completely folded state for indicated buffer conditions. The refolding probability virtually abolished when GLUT3 was embedded in DDM micelles at 0.1 % (w/v), indicating that the lipid bilayer environments provided by the bicelle membranes are essential for inducing the fully folded state.

Source data

Extended Data Fig. 2 Analysis of single-molecule magnetic tweezer data.

a, Precision in determination of the vertical position of a bead as a function of the measurement bandwidth. The plot indicates an ~1 nm resolution when bead positions are averaged over 50 ms (~20 Hz sampling). In our magnetic tweezer experiments, the bicelle phase used for providing the lipid bilayer environments to the target membrane proteins offers additional low-frequency fluctuations, forcing a longer averaging time of 200 ms to achieve the 1 nm accuracy in our membrane protein folding studies. b, Bayesian Information Criteria (BIC) values of WT GLUT3 for each number of states with different bicelle compositions (n = 16 and 11 for 30 mol% and 100 mol% PG, respectively). c, Position of folding intermediates determined by HMM with for different number of states assumed in HMM analysis. The positions of the key intermediates (Uh, If4 (N-domain folded), If6 (C-domain folded) and N) are essentially preserved when the number of assumed states are changed, which only generats additional intermediates either in the middle of either N-domain folding or domain-domain assembly (blue arrows). Thus, our HMM analysis does not randomly assign the intermediate positions out of noisy data, but rather identifies the intermediates implicated in our time-resolved traces in a robust way. d, e, Representative folding traces for WT GLUT3 with 30 mol% PG (d) and 100 mol% PG (e) at 5pN. Two replicates are shown for each condition, and the gray and black traces are 1.2-kHz raw data and 5-Hz median-filtered data, respectively. Red traces indicate the transitions between intermediates identified by HMM.

Extended Data Fig. 3 Sample preparation and folding behavior of S265C/A469C GLUT3.

a, Schematic of the assay using BODIPY-L-cystine. The left panel is the chemical structure of two BODIPY FL fluorophores attached to the amino groups of the disulfide-containing amino acid, cysteine. The right panel shows the structure of GLUT3 before and after the treatment (addition of TCEP or increasing the temperature). Green dots in the right panel are the BODIPY FL fluorophores reacted with cysteines in GLUT3. b, Gel analysis for WT and S265C/A469C GLUT3 in the presence of TCEP. Upper gel shows the amount of GLUT3 stained by Coomassie blue. Lower gel shows the amount of BODIPY FL fluorophores reacted with cysteines in GLUT3. The stained positions are same in both gels. c, Fluorescence profile of BODIPY FL fluorophore-labeled GLUT3 as temperature increased. Dashed lines indicate the melting temperatures of the WT (black) and S265C/A469C GLUT3 (red). Error bars represent SEM (n = 4). d, A mechanical cycle for inducing refolding of a single S265C/A469C GLUT3(GLUT3CC) and corresponding structural states of the protein. e,f, Representative folding traces of GLUT3CC with 30 mol% PG (f) and 100 mol% PG (g) at 5 pN. Definitions of the traces are identical to those of Extended Data Fig. 2d. g, BIC values of GLUT3CC for each number of states (n = 22 and 12 for 30 mol% and 100 mol% PG, respectively).

Source data

Extended Data Fig. 4 Sample preparation and unfolding characteristics of T45C/K115C GLUT3.

a, Atomic contacts among TMHs 1, 2, and 4. Inset shows detailed position of interacting residues (blue for amino group, orange for carboxyl group, and yellow for thiol group). b, The positions of two mutations, T45C/K115C in GLUT3 (GLUT3TM23C). c, An absorbance profile of BODIPY FL fluorophore-labeled GLUT3TM23C as temperature increases. The experiment was done as depicted in Extended Data Fig. 3c. Error bars represent SEM (n = 4). d, Collection of 50Hz-median filtered unfolding traces initiated from N state for GLUT3TM23C. e, Distributions of extension values recorded during high-force unfolding of single GLUT3TM23C proteins. Extension values represent mean ± SD (n = 19).

Extended Data Fig. 5 Determination of folding order for N-domain of GLUT3.

a, Schematic of pulling geometry for N-domain of GLUT3 at 25 pN. dN-domain is the distance between two points of force application before unfolding (PDB: 4ZWC). ∆zi indicates the expected extension increase for GLUT3 for the ith intermediate. zi,p is the extension of the unfolded portion along the membrane for the ith intermediate. di denotes the distance between the points of force application for the ith intermediate. ∆ni is the number of amino acids of the unfolded portion. l is the length of a single amino acid. b, Unfolding extension distribution for the N-domain part of the WT GLUT3 at 25pN. c, Structural information and folding/unfolding order of the N-domain of WT GLUT3. The distance between two orange dashed lines (perpendicular to the membrane) represents the vertical distance between the two points of application (di). This orange dashed line forms an angle of \(\overline {\uptheta}\) with a black dashed line to the unfolded portion of N-domain in the membrane. d, Unfolding extension distribution for the N-domain part of GLUT3TM23C at 25pN. e, Structural information and folding/unfolding order of the N-domain of GLUT3TM23C. The description is the same as (c) except for protein construct. The disulfide bond of GLUT3TM23C did not affect the first unfolding step for N-domain that amounted to ~15.7 nm, confirming that TMHs 5 and 6 constitute the first unfolding step of N-domain. The second unfolding step was slightly reduced to 7.5 nm, which was consistent with the length of last helical turn of the long linker region when TMH 1 would be protected by knotting, mapping the second unfolding step to that of TMH 1 and its linker region. The last two unfolding steps before Uc were reduced to a single step of 4.2 nm, which would reflect unfolding of TMH 4 outside the knotted region. f, Representative traces showing the final unfolding step of a 4.2 nm extension increase for GLUT3TM23C. Three replicates are shown, and the value indicates the distance between two gaussian peaks.

Source data

Extended Data Fig. 6 Symmetrical structure of GLUT3.

a, Structural view of GLUT3’s N-domain with its C2 pseudo-symmetry. b, Structural view and electrostatic potential of the helix triplets composed of TMHs 1, 2, 3 and 4, 5, 6 each. c, Structural view and electrostatic potential of the helix triplets composed of TMHs 1, 5, 6 and 4, 2, 3 each.

Extended Data Fig. 7 EMC preparation and folding characteristics of GLUT3 with β2AR.

a, Structural model of the human ER membrane protein complex (EMC). b, Purified EMC analyzed by SDS-PAGE. The left lane is molecular weight standards and the right lane shows purified EMC. c, Fluorescence profile of EMC before and after freeze-thaw as the temperature is increased. Inset displays the profile for first derivative of fluorescence intensity. d, Representative folding traces of WT (left) and GLUT3CC (right) in the presence of EMC. The definition of each trace is identical to the traces in Extended Data Fig. 2d. e, Purified β2AR analyzed by SDS-PAGE. The left lane is molecular weight standards and the right lane shows β2AR. f, Representative folding traces for single GLUT3 at 5 pN with 30 mol% PG in the bicelles in the presence of 500 nM β2AR. Two replicates are shown. g, Probability distributions of deconvoluted extension values observed under indicated folding conditions at 5pN (n = 11 for the reaction with β2AR). The black and red distributions are revisited from Fig. 4e. The shaded area means SEM. h, BIC values for the indicated number of states (n = 13 and 11 traces for WT GLUT3 and S265C/A469C GLUT3, respectively). i, Positions of folding/unfolding intermediates identified with HMM are depicted for the indicated conditions. Error bars represent SEM (n = 22 and 35 traces for 100 mol% DMPG and 30 mol% DMPG with 500nM EMC, respectively).

Extended Data Fig. 8 Analysis of MD simulation for GLUT3 in lipid bilayer.

a, The average number of contacting water molecule to polar/charged residues in TMHs of N-domain with or without DMPE. Error bars represent SD (n = 4000 for each case). Polar/charged residues in TMHs of GLUT3 for the analysis are as follows. S21, Q23, N27, T28, S71, S78, S80, N98, R124, T135, T156, N158, Q159, T191, Q198, S273, Q277, S279, Q280, Q281, S283, N286, N315, T316, T319, S322, S346, E378, W386, N409, W410, N413. Residues near the GLUT3 pore entries are not chosen which are likely to be exposed to bulk water. b, Interaction frequency of polar/charged residues in N-domain interface with or without DMPE. The value in (a,b) is the average value from 0.6µs to 1.0µs. c, The average number of contacting water molecule to polar/charged residues in TMHs of GLUT3. ‘N’ and ‘C’ represent N, C-domain, respectively. Error bars represent SD (n = 4000 for each case). d, Interaction frequency of polar/charged residues in domain interfaces. The value in (c,d) is the average value from 0.6µs to 1.0µs. e, The average number of contacting water molecule to polar/charged residues in TMHs of GLUT3. Error bars represent SD (n = 2000 for each case). f, Interaction frequency of polar/charged residues in domain interfaces. The value in (e,f) is the average value from 0.3µs to 0.5µs.

Extended Data Fig. 9 Folding characteristics with PE lipid bicelles.

a, Representative folding trace of WT GLUT3 with PE-containing bicelle. Inset shows close-up view of the folding trace. The definition of each trace is identical to the traces in Extended Data Fig. 2d. Two replicates are shown. b, BIC values for the indicated number of states with 15 mol% PE bicelle (n = 11). The intermediate was largely preserved upon addition of DMPE lipids. c, Representative folding trace of WT GLUT3 with PE-containing bicelle in the presence of EMC. d, BIC values for the indicated number of states with 15 mol% PE bicelle in the presence of EMC (n = 10).

Extended Data Fig. 10 Insertion energy of sugar transporters.

a, Insertion energy histogram estimated for TMHs of all sugar porters. b, Insertion energy histogram estimated for C-domain TMHs of metazoan sugar porters. c, Insertion energy histogram estimated for C-domain TMHs of bacteria sugar porters. d, P-values from the Bartlett and Levene tests. 2 sets are used for statistical testing. e, Scatter plot of mean of top 3 insertion energy for N-domain as x-axis and mean of 3 top insertion energy for C-domain as y-axis for sugar transporters. f, Scatter plot of insertion energy variance for N-domain as x-axis and insertion energy variance for C-domain as y-axis. g, Average values of BLOSUM62 score for QLSQQLS motif is calculated for each group. (n = 26, 28, 54 and 24 for bacteria, metazoa, fungi and viridiplantae).

Supplementary information

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Supplementary Figs 1−9.

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Supplementary Data

Unprocessed western blot of Supplementary Fig. 7.

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Source Data Extended Data Fig. 1

Unprocessed gels for Extended Data Fig. 1.

Source Data Extended Data Fig. 3

Unprocessed gels for Extended Data Fig. 3.

Source Data Extended Data Fig. 5

Unprocessed gels for Extended Data Fig. 5.

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Choi, HK., Kang, H., Lee, C. et al. Evolutionary balance between foldability and functionality of a glucose transporter. Nat Chem Biol 18, 713–723 (2022). https://doi.org/10.1038/s41589-022-01002-w

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