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Structure, dynamics and assembly of the ankyrin complex on human red blood cell membrane

Abstract

The cytoskeleton of a red blood cell (RBC) is anchored to the cell membrane by the ankyrin complex. This complex is assembled during RBC genesis and comprises primarily band 3, protein 4.2 and ankyrin, whose mutations contribute to numerous human inherited diseases. High-resolution structures of the ankyrin complex have been long sought-after to understand its assembly and disease-causing mutations. Here, we analyzed native complexes on the human RBC membrane by stepwise fractionation. Cryo-electron microscopy structures of nine band-3-associated complexes reveal that protein 4.2 stabilizes the cytoplasmic domain of band 3 dimer. In turn, the superhelix-shaped ankyrin binds to this protein 4.2 via ankyrin repeats (ARs) 6–13 and to another band 3 dimer via ARs 17–20, bridging two band 3 dimers in the ankyrin complex. Integration of these structures with both prior data and our biochemical data supports a model of ankyrin complex assembly during erythropoiesis and identifies interactions essential for the mechanical stability of RBC.

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Fig. 1: Cryo-EM structures of band 3, protein 4.2 complex and ankyrin-containing complex.
Fig. 2: Protein 4.2 and its interactions with band 3.
Fig. 3: Anchorage of ankyrin to protein 4.2.
Fig. 4: Interaction of ankyrin with band 3.
Fig. 5: Schematic and possible assembly model of the ankyrin complex.

Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-26148 (band 3 dimer), EMD-26145 (B2P1loose), EMD-26146 (B2P1vertical), EMD-26147 (B2P2vertical), EMD-26142 (B2P1diagonal), EMD-26143 (membrane part of B2P1diagonal), EMD-26144 (cytoplasmic part of B2P1diagonal), EMD-26149 (B2P1A1), EMD-26150 (cytoplasmic part of B2P1A1), EMD-26151 (B2P1A2), EMD-26152 (focused refinement of B2P1A2), EMD-26153 (B4P1A1) and EMD-26154 ((B2P1A1)2). Model coordinates have been deposited in the Protein Data Bank under accession numbers 7TW2 (band 3 dimer), 7TW0 (B2P1vertical), 7TW1 (B2P2vertical), 7TVZ (B2P1diagonal), 7TW3 (B2P1A1), 7TW5 (B2P1A2) and 7TW6 (B4P1A1). Other structures used in this study were retrieved from the PDB with accession codes 4YZF for the crystal structure of band 3 membrane domain, 1HYN for the crystal structure of band 3 cytoplasmic domain, 1N11 for ARs 13–24 of ankyrinR, 4RLV for ARs 1–24 of ankyrinB, 1L9N for transglutaminase 3 and 6KI1 for BicA. All other data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. Source data are provided with this paper.

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Acknowledgements

We thank T. Nguyen, J. Zhen and A. Stevens for editorial assistance. This project is supported by grants from the US NIH (R01GM071940 to Z.H.Z.). We acknowledge the use of resources at the Electron Imaging Center for Nanomachines supported by UCLA and grants from the NIH (1S10OD018111 and 1U24GM116792) and the National Science Foundation (DBI-1338135 and DMR-1548924).

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Authors and Affiliations

Authors

Contributions

Z.H.Z. conceived the project. X.X. and S.L. prepared samples and acquired and analyzed cryo-EM data. X.X. engineered and isolated the recombinant proteins and performed biochemistry analyses. S.L. and X.X. built the models. X.X., S.L. and Z.H.Z. interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Z. Hong Zhou.

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The authors declare no competing interests.

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Nature Structural and Molecular Biology thanks Ash Toye, Yifan Cheng, and Werner KÃhlbrandt for their contribution to the peer review of this work. Primary Handling editor: Florian Ullrich, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Purification and cryo-EM reconstruction of the erythrocyte membrane proteins.

(a) Workflow of the stepwise fractionation of erythrocyte membrane proteins. (b) The second gel-filtration chromatography profile of the low-salt fraction. The result from SDS-PAGE analysis of the peak fractions is inserted in the upper left corner. The peak fractions were applied to SDS-PAGE and visualized by Coomassie blue staining. Dashed blue box on the gel and blue bar on the chromatogram indicate the fractions collected for cryo-EM. (c) The first gel-filtration chromatography profile of the high-salt fraction. Green and magenta boxes on the gel and green and magenta bars on the chromatogram indicate the fractions collected for the protein 4.2 complex and ankyrin complex, respectively. (d) The second gel-filtration chromatography profile and corresponding gel of the protein 4.2 complex. Dashed green box on the gel and green bar on the chromatogram indicate the fractions collected for cryo-EM. (e) The gel-filtration chromatography profile of the ankyrin complex after Grafix purification. Magenta bar on the chromatogram indicates the fractions collected for cryo-EM.

Extended Data Fig. 2 Cryo-EM analysis of the low-salt fraction (band 3).

(a) Representative cryo-EM image of the low-salt fraction from 9455 images collected. (b) Selected 2D class averages of the cryo-EM particle images. (c) Flow chart of cryo-EM data processing. (d) Gold-standard Fourier shell correlation (FSC) curve for 3D reconstruction. (e) Angular distribution of cryo-EM reconstructions used for final refinement. (f) Density of the membrane domain. (g) Atomic model of band 3 cytoplasmic domain fitted into the cryo-EM density. A lower map threshold is used in (g) compared to that of (f) to better present the cytoplasmic domain.

Extended Data Fig. 3 Image processing for the cryo-EM data of the high-salt fraction 1 (band 3-protein 4.2 complex).

(a) Representative cryo-EM image of the high-salt fraction 1 from 20842 images collected. (b) Selected 2D class averages of the cryo-EM particle images. (c) Flow chart of cryo-EM data processing. (d), Gold-standard Fourier shell correlation (FSC) curves for 3D reconstructions. (e) Local resolution of the overall map of B2P1diagonal complex. (f) Angular distribution of cryo-EM reconstruction of B2P1diagonal complex used for final refinement. (g-h) Local resolutions of the focused refinement maps of the cytoplasmic part and membrane part of B2P1diagonal complex. (i) Representative cryo-EM density maps of the B2P1diagonal complex.

Extended Data Fig. 4 Cryo-EM analysis of the high-salt fraction 2 (ankyrin complex).

(a) Representative cryo-EM image of the high-salt fraction 2 from 21187 images collected. (b) Selected 2D class averages of cryo-EM particle images. (c) Flow chart of cryo-EM data processing. (d) Gold-standard Fourier shell correlation (FSC) curves for 3D reconstructions. (e) Local resolution of the overall map of B2P1A1 complex. (f) Angular distribution of cryo-EM reconstruction of B2P1A1 complex. (g) Representative cryo-EM density maps of the B2P1A1 complex showing the fragments of ankyrin and protein 4.2 at their binding interface. (h) Representative cryo-EM density maps of the B2P1A2 complex showing the fragments of ankyrin and band 3 at their binding interface.

Extended Data Fig. 5 Structural analysis of the band 3 membrane domain.

(a) Superposition of the band 3 membrane domain in B2P1diagonal complex and reported crystal structure (PDB: 4YZF)16. (b) Topology of the transmembrane helices of band 3. (c) Density of the DDM molecule at the interface of the core and gate domain. (d) Enlarged view of the substrate binding site in B2P1diagonal complex. Four water molecules were tentatively modelled into the cryo-EM density of band 3 near the substrate binding site. (e) Comparison of the substrate binding site in band 3 with that in bicarbonate transporter BicA (PDB: 6KI1)25.

Extended Data Fig. 6 Sequence alignment of band 3 from different species.

Sequences of human band 3 (P02730), mouse band 3 (P04919), rabbit band 3 (G1SLY0), bovine band 3 (Q9XSW5), horse band 3 (Q2Z1P9), chicken band 3 (P15575), frog band 3 (F6XSL8) and fish band 3 (Q7ZZJ7). The sequence alignment is done using the Clustal Omega server72; the figure is generated by ESPript 373. Cyan triangles represent the band 3 residues interacting with protein 4.2; magenta bars indicate the regions of band 3 interacting with ankyrin; reported disease mutations on human band 3 are labeled as red circles.

Extended Data Fig. 7 Structure of protein 4.2.

(a) Structure of the core domain shown in ribbon. Residue numbers of its N and C terminus are labeled. (b) Superposition of protein 4.2 with transglutaminase (gray, PDB: 1L9N)31, showing the missing catalytic triad in protein 4.2. (c-e) Structures of the three Ig-like domains and illustrations of their secondary structure. (f) The electrostatic surface of protein 4.2, showing its membrane binding site (blue dashed circle) and band 3 binding interface (orange dashed circle). (g-h) Sequence conservation of protein 4.2 among mammals mapped to the structure. Orientation in (g) is the same as that in (f). Orange dashed circle shows the band 3 binding interface; magenta dashed box shows the ankyrin binding interface.

Extended Data Fig. 8 Sequence alignment of protein 4.2 from different species.

Sequences of human protein 4.2 (P16452), mouse protein 4.2 (P49222), rabbit protein (G1TDR3), bovine protein 4.2 (O46510), horse protein 4.2 (F6ZDW1), chicken protein 4.2 (E1BQZ4), frog protein 4.2 (XP_018090678.1) and human transglutaminase 3 (Q08188). The sequence alignment is done using the Clustal Omega server72; the figure is generated in ESPript 373. Salmon triangles represent protein 4.2 residues interacting with band 3; magenta bars indicate the regions of protein 4.2 interacting with ankyrin; reported disease mutations on human protein 4.2 are labeled as red circles; black stars indicate the catalytic residues of human transglutaminase 3.

Extended Data Fig. 9 Conformational changes of band 3 and protein 4.2 during the assembly process.

(a, b) Density map of the B2P1vertical complex and B2P1diagonal complex. Red box indicates the anchorage site of protein 4.2 N-termini to the membrane. (c) Rotation of protein 4.2 (red arrow) from vertical (transparent grey surface) to diagonal conformation (green surface). The two complexes are superposed according to the membrane domains of band 3. The cytoplasmic domains of band 3 are omitted for clarity. Angles between the membrane (grey bar) and protein 4.2 are labeled. (d-e) Rotation of the cytoplasmic domain of band 3 (red arrow) from B2P1vertical to B2P1diagonal complex viewed from the cytoplasmic side. The membrane domain of band 3 is shown as transparent surface and cytoplasmic domain as ribbon. Protein 4.2 is indicated as a green oval for clarity. (f) Density map of the B2P1diagonal complex sharpened with B-factor of −50 Å2 showing the interaction of the CM-linker with protein 4.2. (g-h) Ribbon representation of the CM-linker region. Residues of protein 4.2 interacting with the CM-linker are labeled.

Extended Data Fig. 10 Analysis of the ankyrin-containing complexes.

(a) Atomic model of B4P1A1 complex in ribbon superposed with the density map at a low threshold. Red arrow indicates the density of the membrane domain of the second band 3 dimer. (b) Analytical gel filtration assay showing the assembly of B4P1A1 complex in vitro. Dashed boxes show the position of B4P1A1 complex. Experiments were repeated for two times with similar results. (c) Atomic model of B2P1A2 complex in ribbon superposed with the density map at a low threshold. The density for the second band 3 dimer is indicated by arrow. (d) Analytical gel filtration assay showing the assembly of B2P1A2 complex in vitro. Dashed boxes show the position of B2P1A2 complex. The gel-filtration and SDS-PAGE results of protein 4.2 complex, band 3 cytoplasmic domain and ARs 1–24 are the same as that in (b). Second band 3 dimer may be incorporated into this complex, resulting in the B4P1A2. Experiments were repeated for two times with similar results. (e) Atomic model of (B2P1A1)2 complex in ribbon superposed with the density map. Boxes show the position of individual B2P1A1 complexes. (f) Reported disease mutations mapped on the structure of B4P1A1 complex.

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Supplementary Video 1

Sequential assembly of the ankyrin complex

Source data

Source Data Fig. 4

Uncropped gels for Fig. 4c

Source Data Extended Data Fig. 10

Uncropped gels for Extended Data Fig. 10

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Xia, X., Liu, S. & Zhou, Z.H. Structure, dynamics and assembly of the ankyrin complex on human red blood cell membrane. Nat Struct Mol Biol 29, 698–705 (2022). https://doi.org/10.1038/s41594-022-00779-7

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