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Native DGC structure rationalizes muscular dystrophy-causing mutations

Nature volume 637, pages 1261–1271 (2025)Cite this article

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Abstract

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder marked by progressive muscle wasting leading to premature mortality1,2. Discovery of the DMD gene encoding dystrophin both revealed the cause of DMD and helped identify a family of at least ten dystrophin-associated proteins at the muscle cell membrane, collectively forming the dystrophin–glycoprotein complex (DGC)3,4,5,6,7,8,9. The DGC links the extracellular matrix to the cytoskeleton, but, despite its importance, its molecular architecture has remained elusive. Here we determined the native cryo-electron microscopy structure of rabbit DGC and conducted biochemical analyses to reveal its intricate molecular configuration. An unexpected β-helix comprising β-, γ- and δ-sarcoglycan forms an extracellular platform that interacts with α-dystroglycan, β-dystroglycan and α-sarcoglycan, allowing α-dystroglycan to contact the extracellular matrix. In the membrane, sarcospan anchors β-dystroglycan to the β-, γ- and δ-sarcoglycan trimer, while in the cytoplasm, β-dystroglycan’s juxtamembrane fragment binds dystrophin’s ZZ domain. Through these interactions, the DGC links laminin 2 to intracellular actin. Additionally, dystrophin’s WW domain, along with its EF-hand 1 domain, interacts with α-dystrobrevin. A disease-causing mutation mapping to the WW domain weakens this interaction, as confirmed by deletion of the WW domain in biochemical assays. Our findings rationalize more than 110 mutations affecting single residues associated with various muscular dystrophy subtypes and contribute to ongoing therapeutic developments, including protein restoration, upregulation of compensatory genes and gene replacement.

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Fig. 1: Enrichment and overall architecture of the native DGC from rabbit skeletal muscle.
Fig. 2: Triple β-helix structure of β-SG–γ-SG–δ-SG and its interaction with α-SG.
Fig. 3: Architecture and interactions of the DG complex with SGs.
Fig. 4: Sarcospan-mediated linkage of β-DG to SGs.
Fig. 5: Dystrophin and α-dystrobrevin dimerize through their cysteine-rich regions.
Fig. 6: Model of the DGC on the sarcolemma.

Data availability

The cryo-EM maps and atomic coordinates have been deposited to the EMDB (EMD-45165) and PDB (9C3C) databases, respectively. Other structures used in this study were retrieved from the PDB with accession codes 6DLH for endo-fucoidan hydrolase MfFcnA4 and 1EG4 for dystrophin’s WW and EF-hand domains with the PPXY peptide of β-DG. The gene IDs of the DGC components were obtained from NCBI: 100009208 for β-SG, 100009214 for γ-SG, 100351233 for δ-SG, 100009178 for α-SG, 100009278 for DG, 100355731 for dystrophin and 100351412 for α-dystrobrevin. All other data are available within the main text or the Extended Data. Raw data and source images are available in the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank R. H. Crosbie for comments on the scientific content and T. Nguyen, J. Jih and L. Wang for comments on the writing and presentation of the paper. This project is supported by a grant from the US National Institutes of Health (R01GM071940 to Z.H.Z.). We acknowledge the use of resources at the Electron Imaging Center for NanoSystems, supported by UCLA and grants from the National Institutes of Health (S10RR23057 and S10OD018111) and National Science Foundation (DBI-1338135 and DMR-1548924).

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Author notes

  1. These authors contributed equally: Shiheng Liu, Tiantian Su, Xian Xia

Authors and Affiliations

  1. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Shiheng Liu, Tiantian Su, Xian Xia & Z. Hong Zhou

  2. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Shiheng Liu, Tiantian Su, Xian Xia & Z. Hong Zhou

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  1. Shiheng Liu
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  2. Tiantian Su
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Contributions

Z.H.Z. conceived the project and supervised the research. S.L. designed the experimental protocols. S.L. and X.X. prepared samples and collected cryo-EM images. S.L., T.S. and X.X. determined the 3D structures. S.L. and T.S. built atomic models and generated the figures. T.S. and S.L. engineered the recombinant proteins and performed biochemical analyses. S.L. and T.S. interpreted the results, prepared the illustrations and wrote the original draft of the manuscript. S.L., T.S., X.X. and Z.H.Z. contributed to the editing of the manuscript.

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Correspondence to Z. Hong Zhou.

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Extended data figures and tables

Extended Data Fig. 1 Representative cryo-EM images and 2D analyses.

a,b,Negative staining (a) and drift-corrected cryo-EM (b) micrographs of DGC isolated from rabbit skeletal muscle. Representative side view and top view particles are shown in red and yellow circles, respectively. c, Representative 2D class averages of DGC isolate. Top views and side views of the DGC are labelled with red and yellow boxes, respectively.

Extended Data Fig. 2 Cryo-EM structural determination of DGC.

a, Data processing workflow. Binned 2 (pixel size of 2.72 Å) and binned 1 (pixel size of 1.36 Å, grey shaded) data processing is separated via dashed line. The masks used in focused 3D classification are outlined with coloured dashed lines. Data processing in RELION and cryoSPARC is denoted by a circular inscribed “R” and “C”, respectively. See Methods for more details. b, FSC as a function of spatial frequency demonstrating the resolution of the final reconstruction of DGC. c,d, View direction distribution histogram (c) and posterior precision plot (d) show view diversity of all particles used for the final map of DGC (from cryoSPARC). e, Local resolution estimation (from cryoSPARC) using a local FSC threshold of 0.5. f, FSC coefficients as a functional of spatial frequency between model and cryo-EM density maps. The generally similar appearances between the FSC curves obtained with half maps with (red) and without (blue) model refinement indicate that the refinement of the atomic coordinates did not suffer from severe over-fitting.

Source Data

Extended Data Fig. 3 Representative cryo-EM density maps of DGC.

C-terminal cap of β-, δ-, γ-SG in panel a and dystrophin (EF-hands+WW)–dystrobrevin (EF-hands) heterodimer in panel f shown using the unsharpened 4.3 Å map. CDHL1-SEA1 of α-SG (d) displayed using a gaussian low-pass filtered map. All others are shown using sharpened densities of the 4.1-Å local-refined map or the 4.3-Å map.

Extended Data Fig. 4 Structure prediction of the triplex structure of β-, γ- or δ-SG using artificial intelligence (AI) programs.

Panels from left to right: cryo-EM model of the β-SG–γ-SG–δ-SG trimer (panel 1) and the predicted models of single-component trimers (panels 2-4). All predicted models were generated using AlphaFold. Regions with pLDDT > 90 are expected to be modelled to high accuracy; regions with pLDDT between 70 and 90 are expected to be modelled well; regions with pLDDT between 50 and 70 are modelled with low confidence and should be treated with caution.

Extended Data Fig. 5 Transfected β-, γ- and δ-SG separated by SDS-PAGE and visualized by immunoblot with Myc tag-specific antibody.

For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6 Identifying the potential calcium (Ca2+) binding site in DGC’s CDHL domains.

a, Superposition of the CDHL domains with focused views of the Ca2+ coordination site in endo-fucoidan hydrolase MfFcnA4 (PDB ID: 6DLH, purple), and potential Ca2+ binding sites in dystroglycans (salmon and grey) and α-sarcoglycan (cyan). Residues possibly involved in Ca2+ coordination are annotated. b, Cross-species multiple sequence alignment of the three CDHL domains in DGC. Red background depicts identical residues in all CDHL domains. Conserved residues are coloured in red and divergent residues are in black. All potential coordination residues shown in a are labelled with red circles. MfFcnA4 residues structurally homologous to CDHL2 positions are numbered; green-filled dash circles (half: not conserved; full: identical) indicate potential Ca2+ binding sites in CDHL2.

Extended Data Fig. 7 Divergent domain orientations between CDHL and SEA in the three CDHL-SEA modules of DGC.

a, Structural superposition of the three CDHL-SEA modules within DGC. SEA-CDHL modules are aligned against the SEA domain. b, Unique orientations are essential for the TM-proximal loop of β-DG (left), α-DG (top right) and α-SG (bottom right) to interact with the β-helix. The circle slash denotes interaction disruption by detachment, while the circle cross indicates interaction disruption via atomic clashes.

Extended Data Fig. 8 Comparison of β-DG binding sarcospan with both partner-binding and apo states of the canonical tetraspanin CD81.

Sarcospan and tetraspanin CD81 are depicted as ribbons, while β-DG and CD81’s binding partner are shown as surfaces. CD81’s unmodelled regions are represented by dashed lines.

Extended Data Fig. 9 Subcellular localization of dystrophin cysteine-rich region and its WW deletion construct.

The fragments used are residues 3042-3426 and 3077-3426 for dystrophin and dystrophin (ΔWW), respectively.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Fig. 1

Uncropped gels used in the preparation of Figs. 1b, 2g, 3h,i and 5e and Extended Data Fig. 5. Page 1 presents the uncropped SDS–PAGE gel for Fig. 1b; pages 2–6 contain uncropped western blot images from the co-immunoprecipitation assays used for Figs. 2g, 3h,i and 5e and Extended Data Fig. 5. Black dashed boxes indicate the cropped areas displayed in the respective figures.

Reporting Summary

Supplementary Table 1

Pathogenic mutations in DGs, dystrophin and α-dystrobrevin. Mutation residues listed in the third column are numbered according to the sequences of the human proteins. The mutation impacts in bold in the fifth column are inferred from our structural analysis, while the impacts in regular text in the same column are derived from previous reports.

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Liu, S., Su, T., Xia, X. et al. Native DGC structure rationalizes muscular dystrophy-causing mutations. Nature 637, 1261–1271 (2025). https://doi.org/10.1038/s41586-024-08324-w

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