Abstract
Mitochondrial outer membrane proteins (β-OMPs) serve as the first line of communication with the cellular milieu. A crucial β-OMP, the sorting and assembly machinery Sam50, is a 16-stranded transmembrane β-barrel highly conserved in all eukaryotes. Sam50 dysregulation is lethal; yet, molecular elements that regulate Sam50 remain poorly understood. Here, we identify and characterize residues that regulate Sam50 structure and function. Using single-molecule electrophysiology, in vivo function, and stability measurements, we demonstrate that the POlypeptide-TRansport Associated domain is dispensable for Sam50. Complete characterization of the folding mechanism using 165 Xaa→Ala substitutions reveals that Sam50 folds through parallel pathways, with at least two transition states. The folding nucleus is towards the N-terminus, whereas frustrated folding at the C-terminal region kinetically traps the structure. We correlate this unexpected folding of Sam50 with its SAM-assisted assembly. Additionally, our per-residue stability measurements show that destabilizing hotspots in Sam50 are linked to its gating function. Our findings suggest how the dynamic structure of Sam50 offers a functional advantage, with specific residues that regulate folding, stability, and function, also determine the protein’s sensitivity to mutations.
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The source data underlying Figs. 1c, 1e, 1i, 2a, 2c, and 4a, and other SI images are provided as a Source Data file. Accession codes and hyperlinks for structures from PDB: 7BTW; 7BTY; 7VKU. Should any raw data files be needed in another format, they are available from the corresponding author. Source data are provided in this paper.
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Source files and scripts pertaining to the MD simulations are provided as a separate supplementary file, and contain the initial and final system coordinates (.PDB), lipid and force-field parameter files (.itp) and system topology files (.top), bfactor-mapped.pdb derived from per-residue fluctuations, as well as RMSD, per-residue RMSF, free energy landscape data, and distance analysis data (.txt,.xvg), are available in the Source Data file.
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Acknowledgements
We thank Prof. Dr. Doron Rapaport (University of Tuebingen, Germany) for the S. cerevisiae SAM50 gene. We thank Prof. Dr. Nils Wiedemann (University of Freiburg, Germany) and Prof. Dr. Bernard Guiard (Center de Génétique Moléculaire, CNRS, France) for S. cerevisiae YPH499 sam50::ade2 (sam50Δ shuffle strain) and Prof. Dr. Wiedemann for the pFL39-SAM50 E. coli-yeast shuttle vector. P.B. Tiwari generated pET3a-SAM50Δ120 from the FL variant. R.R. and J.K. thank the University Grants Commission, and AR thanks the Prime Minister’s Research Fellowship, Govt. of India, for a research fellowship. R.M. is a Wellcome Trust-DBT India Alliance Senior Fellow. This work was supported primarily by the Wellcome Trust-Department of Biotechnology India Alliance grant IA/S/20/2/505182, and in part by the Science and Engineering Research Board grant SPR/2021/000018 and Department of Biotechnology grant BT/PR28858/BRB/10/1718/2018, to R.M.
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R.M.: research design, funding, study supervision, interpretation of results, and manuscript writing. R.R.: library preparation (DNA and protein), electrophysiology measurements, yeast experiments, equilibrium titrations, kinetics measurements, MD simulations, data analysis, and interpretation of results. S.G. and J.K.: library preparation (DNA and protein), equilibrium titrations, data analysis. A.R.: electrophysiology measurements. MD simulations, and associated data analysis. All authors have approved the manuscript contents.
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Ravi, R., Gupta, S., Kumari, J. et al. Linking multipoint folding and stability with functional regulation in the mitochondrial transmembrane β-barrel Sam50. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70904-3
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DOI: https://doi.org/10.1038/s41467-026-70904-3
