Abstract
The Pyrococcus horikoshii amino acid transporter GltPh revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFSopen-1, to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on GltPh are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology.
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Data availability
All data supporting the findings of this manuscript are available within the article, supplementary information files and source data file, provided with this paper. Additional information and raw data are available from the corresponding author upon reasonable request. PDB structures used in this article are available in the PDB with the following access codes: 6X17, 6X12, 4OYE, 4P19, 6WZB and 6WYK. The reporting summary for this article is available as a supplementary information file. Source data are provided with this paper.
Code availability
Codes used for HS-AFM single-molecule structural analysis and LAFM map construction are available on GitHub (https://github.com/rafaeljiang23/SingleMoleculeStructuralBiology (ref. 57)).
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Acknowledgements
We thank J. S. Dittman for important discussions. We thank M. Imamura, Y. Pan and E. Shin for the application of MEMPR to different membrane protein–lipid systems. Funding: this work was funded by grants from the National Institutes of Health, National Center for Complementary and Integrative Health (NCCIH), grant no. DP1AT010874 (Scheuring), and National Institute of Neurological Disorders and Stroke (NINDS), grant no. R01NS110790 (Scheuring).
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Contributions
Y.J. and S.S. designed the study. Y.J. performed all HS-AFM experiments. A.M. performed HS-AFM developments and optimized HS-AFM performance. Y.J. and S.S. analyzed HS-AFM data. X.W. purified GltPh. B.Q. purified hEAAT3g. Y.J., O.B. and S.S. wrote the manuscript. S.S. supervised the project.
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Nature Structural & Molecular Biology thanks Dorothy Erie, Albert Guskov and Melanie Köhler for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.
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Extended data
Extended Data Fig. 1 GltPh PDB structures.
(a), (b) and (c) PDB structures of GltPh outward-facing state open (OFS open, light blue, PDB 6X17), outward-facing state closed (OFS closed, dark blue, PDB 4OYE), inward-facing state open (IFS open, yellow, PDB 6X12), and inward-facing state closed (IFS closed, green, PDB 4P19) states: (a) Transport domain (blue) and trimerization domain (gray). (b) Superimposed open and closed structures of the OFS (top) and IFS (bottom) GltPh. Large domain movements present in the IFS structures. Inset: positions of the hairpin 2 (HP2), showing the open and closed ligand binding pocket. (c) Surface representations of the structures from the extracellular side (top), in a side view (middle), and from the cytoplasmic side (bottom). Black arrowheads: Trimerization domain height levels on the cytoplasmic side. Red arrowheads: Transport domain height levels on the cytoplasmic side. The height difference estimates indicate the height protrusion of the transport domain relative to the trimerization domain, viewed from the cytoplasmic side.
Extended Data Fig. 2 Negative stain electron microscopy (EM) of GltPh reconstitution.
(a) Electron micrograph of proteo-liposomes following reconstitution at low lipid-to-protein ratio (LPR) of 0.7 (w:w), step 1 in the MEMPR method (Fig. 1, step 1). (b) Zoom-in of dashed outlined region in (a). Note the graininess of the vesicles characteristic of densely packed proteo-liposomes. Similar results were obtained in all samples. White arrowheads: Proteo-liposomes. Consistent results were obtained from > 3 reconstitution samples.
Extended Data Fig. 3 Overview HS-AFM imaging of the GltPh reconstitution.
(a), (b), and (c). HS-AFM images of membranes with densely packed GltPh molecules at step 1 in the MEMPR method (Fig. 1, step 1). GltPh molecules coverage of the membrane is estimated as ~ 90% in (a), ~ 80% in (b), and ~ 90% in (c). Red circles: Discernable GltPh trimers in the densely packed GltPh clusters. Consistent results were obtained from > 3 HS-AFM experiments and > 3 reconstitution samples.
Extended Data Fig. 4 Negative stain electron microscopy (EM) of GltPh pre-physisorption mixture.
(a) Micrograph with 2D-sheets following the mixture of reconstituted proteo-liposomes with empty liposomes at a buffer contained 1x CMC detergent (DDM), at step 3 in the MEMPR method (Fig. 1, step 3). (b) Zoom-in of the dashed outlined region in (a). Similar results were obtained in all samples. White arrowheads: Open membrane-sheets after mixing reconstituted GltPh proteo-liposomes (Extended Data Fig. 2) and empty SUVs. Consistent results were obtained from > 3 reconstitution samples.
Extended Data Fig. 5 Droplet angle method for the analysis of residual detergent in the sample at different sample preparation stages.
Top left: The initial pre-physisorption mixture contains 1x CMC DDM (see main text Fig. 1, step 3). Panels 2 to 6: Buffer exchange results in ~ 1/32x CMC DDM (see main text Fig. 1, step 4). Panel 7: Inserting the sample stage into the detergent free imaging buffer in the HS-AFM fluid cell further dilutes the remaining detergent to ~ 1/1500x CMC DDM. For comparison a droplet of the detergent free buffer solution without DDM (last panel).
Extended Data Fig. 6 HS-AFM imaging of freely diffusing GltPh in extended lipid bilayers on bare mica.
(a), (b) and (c). HS-AFM frames of non-immobilized GltPh molecules in extended lipid bilayers, on the freshly cleaved mica without any pretreatment, step 5a of the MEMPR method (Fig. 1, step 5a). Freely diffusing (a) and cluster-forming (b and c) GtlPh molecules were observed in different regions of the continuous membrane (Supplementary Videos 1–3). Consistent results were obtained from > 3 HS-AFM experiments and > 3 reconstitution samples.
Extended Data Fig. 7 HS-AFM imaging of immobilized GltPh in extended lipid bilayers on poly-Lys pretreated mica.
(a), (b) and (c). HS-AFM frames of immobilized GltPh molecules in extended lipid bilayers, on the freshly cleaved mica with pretreatment of poly-Lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b). GltPh molecule coverage of the membrane is estimated as ~ 30% in (a), and ~ 10% in (b) and (c) (Supplementary Videos 4–6). White arrowheads: a GltPh cluster showing lateral dynamics (see main text). Consistent results were obtained from > 3 HS-AFM experiments and > 3 reconstitution samples.
Extended Data Fig. 8 Two-state conformation-time trace of a GltPh protomer showing different kinetic modes.
(a) Selected HS-AFM frames (top) and corresponding states of individual protomers (bottom) of an isolated, immobilized GltPh trimer in an extended lipid bilayer viewed from the cytoplasmic side. (b) Two-state (OFS and IFS) conformation-time trace of an individual protomer. Color coding in (a) and (b): OFS (blue) and IFS (grey). Protomer p2 switches kinetic modes: Mode 1: ~ 20 s inactive in IFS. Mode 2: ~ 40 s inactive in OFS. Mode 3: ~ 30 s highly active mode with frequent OFS-IFS transitions (Supplementary Video 12). Two-state conformation-time traces showing different kinetic modes were observed in > 3 single molecules from different reconstitution samples.
Extended Data Fig. 9 HS-AFM fast imaging (50 frames per second) of an immobilized GltPh in extended lipid bilayers.
HS-AFM frames of an isolated and immobilized GltPh trimer in extended lipid bilayers, viewed from the cytoplasmic side on the freshly cleaved mica with pretreatment of poly-Lys (Fig. 1, step 5b). The MEMPR method enables fast imaging of single immobilized molecules with unprecedented resolutions, 0.02 s/frame and 0.25 nm/pixel, and stability, for ~ 80 s in this example (Supplementary Video 13).
Extended Data Fig. 10 PDB Structure of a Cl− Conducting State (ClCS) GltPh.
PDB structures of GltPh IFS (XL3, gray, PDB 6WZB), Cl−conducting state (ClCS, marron, PDB 6WYK), IFS closed (green, PDB 4P19, see Extended Data Fig. 1), and IFS open (green, PDB 6X12, see Extended Data Fig. 1). Top: Superimposed structures of IFS XL3 and ClCS (left), and of IFS open and IFS closed (right). Middle: Superimposed structures of IFS open and ClCS (left), and of IFS closed and IFS XL3 (right). Bottom: Superimposed structures of IFS closed and ClCS (left), and of IFS open and IFS XL3 (right). These comparisons structurally relate ClCS to IFS open and IFS XL3 to IFS closed. IFS closed and IFS XL3 structures were collected in the apo condition. IFS open structure was collected in the presence of DL-threo-β-benzyloxyaspartate (TBOA), and ClCS in the presence of Na+ and Asp (transport condition).
Supplementary information
Supplementary Information
Supplementary Note, Figs. 1–3 and Table 1.
Supplementary Video 1
HS-AFM video of freely diffusing GltPh in extended lipid bilayers on freshly cleaved mica. HS-AFM video of diffusing GltPh trimers in extended lipid bilayers, on a freshly cleaved mica, at step 5a of the MEMPR method (Fig. 1, step 5a, and Extended Data Fig. 6a). Imaging parameters were 2 frames per s, 1 nm per pixel.
Supplementary Video 2
HS-AFM video of cluster-forming GltPh in extended lipid bilayers on freshly cleaved mica area 1. HS-AFM video of cluster-forming GltPh molecules in extended lipid bilayers, on a freshly cleaved mica, at step 5a of the MEMPR method (Fig. 1, step 5a, and Extended Data Fig. 6b). Imaging parameters were 2 frames per s, 1 nm per pixel.
Supplementary Video 3
HS-AFM video of cluster-forming GltPh in extended lipid bilayers on freshly cleaved mica area 2. HS-AFM video of cluster-forming GltPh molecules in extended lipid bilayers, on a freshly cleaved mica, at step 5a of the MEMPR method (Fig. 1, step 5a, and Extended Data Fig. 6c). Imaging parameters were 2 frames per s, 1 nm per pixel.
Supplementary Video 4
HS-AFM video of immobilized GltPh in extended lipid bilayers on poly-lys pretreated mica area 1. HS-AFM video of immobilized GltPh molecules in extended lipid bilayers, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, Fig. 2b and Extended Data Fig. 7a). Imaging parameters were 2 frames per s, 1 nm per pixel.
Supplementary Video 5
HS-AFM video of immobilized GltPh in extended lipid bilayers on poly-lys pretreated mica area 2. HS-AFM video of immobilized GltPh molecules in extended lipid bilayers, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Extended Data Fig. 7b). Imaging parameters were 2 frames per s, 1 nm per pixel.
Supplementary Video 6
HS-AFM video of immobilized GltPh in extended lipid bilayers on poly-lys pretreated mica area 2. HS-AFM video of immobilized GltPh molecules in extended lipid bilayers, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Extended Data Fig. 7c). Imaging parameters were 2 frames per s, 1 nm per pixel.
Supplementary Video 7
HS-AFM video of an individual GltPh trimer viewed from the extracellular side 1. HS-AFM video of an immobilized GltPh molecule viewed from the extracellular side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Fig. 2c). Imaging parameters were 10 frames per s, 0.25 nm per pixel.
Supplementary Video 8
HS-AFM video of an individual GltPh trimer viewed from the extracellular side 2. HS-AFM video of an immobilized GltPh molecule viewed from the extracellular side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Fig. 2d). Imaging parameters were 10 frames per s, 0.25 nm per pixel.
Supplementary Video 9
HS-AFM video of an individual GltPh trimer viewed from the cytoplasmic side 1. HS-AFM video of an immobilized GltPh molecule viewed from the extracellular side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Fig. 2e). Imaging parameters were 10 frames per s, 0.25 nm per pixel.
Supplementary Video 10
HS-AFM video of an individual GltPh trimer viewed from the cytoplasmic side 2. HS-AFM video of an immobilized GltPh molecule viewed from the extracellular side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Fig. 2f). Imaging parameters were 10 frames per s, 0.25 nm per pixel.
Supplementary Video 11
HS-AFM video of a dimer of GltPh trimers. HS-AFM video of a dimer of GltPh trimer viewed from the extracellular side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Fig. 2g). Imaging parameters were 10 frames per s, 0.5 nm per pixel.
Supplementary Video 12
HS-AFM video of a GltPh protomer showing different kinetic modes. HS-AFM video of an immobilized GltPh trimer viewed from the cytoplasmic side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Extended Data Fig. 8). One protomer displayed switching modes: mode 1, ~20 s inactive in IFS; mode 2, ~40 s inactive in OFS; and mode 3, ~30 s highly active mode with frequent OFS–IFS transitions. Imaging parameters were 10 frames per s, 0.25 nm per pixel.
Supplementary Video 13
HS-AFM fast imaging of an individual GltPh trimer. HS-AFM video of an immobilized GltPh trimer viewed from the cytoplasmic side in an extended lipid bilayer, on the freshly cleaved mica with pretreatment of poly-lys (coated mica), at step 5b of the MEMPR method (Fig. 1, step 5b, and Extended Data Fig. 9). The MEMPR method enables fast imaging of single immobilized molecules with resolutions of 0.02 s per frame and 0.25 nm s−1, and stability, for ~80 s in this example.
Supplementary Data 1
Source data for Supplementary Fig. 3.
Source data
Source Data Figs. 3–6 and Extended Data Fig. 8
Statistical source data.
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Jiang, Y., Miyagi, A., Wang, X. et al. HS-AFM single-molecule structural biology uncovers basis of transporter wanderlust kinetics. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01260-3
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DOI: https://doi.org/10.1038/s41594-024-01260-3
