Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Mitochondrial Sam50 β-Barrel: Folding Pathways and Functional Regulators (#132)

Roshika Ravi 1 , Swadha Gupta 1 , Jyoti Kumari 1 , Radhakrishnan Mahalakshmi 1
  1. Molecular Biophysics Laboratory, Department of Biological Sciences , Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, India

The mitochondrial outer membrane possess unique β-barrel membrane protein channels, the biogenesis of which requires the highly conserved sorting and assembly machinery (SAM).[1,2] The core subunit of the SAM complex, Sam50, is a 16-stranded β-barrel with ancestral homology to the bacterial assembly machinery. Cryo-EM studies of Sam50[1,2] have provided structural insights into the intermediates formed during SAM–assisted folding and release of the nascent β-barrel. Yet, we lack molecular information on microstates formed during Sam50 assembly, per-residue contributions to the end-state stability, role of the membrane in β-barrel folding, and whether a structure–function trade-off exists for mitochondrial membrane proteins. Most importantly, deducing how the primary sequence dictates Sam50 folding has direct implications in our understanding of β-barrel misfolding and aggregation, and the onset of neurodegenerative diseases.[3] Particularly with membrane proteins, the transmembrane region, which spans the bilayer, is a critical determining element for folding vs aggregation. Here, we characterized the contribution of the transmembrane domain of Sam50 (Sam50Δ120) in its folding kinetics, thermodynamic stability, and in vivo function of this essential protein. Folding kinetics in membrane vesicles reveals parallel pathways for Sam50Δ120 assembly. Transition state structures in both pathways (Φ-value analysis) reveal that C-terminal strands exhibit frustrated folding forming non-native contacts, and requiring chaperone assistance in vivo. The β-barrel N-terminal zone assembles spontaneously; however, it lowers the post-folding stability of Sam50Δ120, thereby forming a metastable scaffold that is essential for its chaperone function. Residues demonstrating frustrated folding are functionally significant in yeast survival. Our studies bridge global Sam50–dependent mechanisms of β-barrel folding with microscopic regulatory elements vital for the assembly and function of the core Sam50 β-barrel itself. Further elucidation of early intermediates in Sam50 folding and correlation with in vivo function will provide mechanistic details of mitochondrial biogenesis at the atomic level.

  1. Takeda, H., et al., Mitochondrial sorting and assembly machinery operates by β-barrel switching. Nature. (2021), 590(7844): 163-169.
  2. Wang Q, et al., Structural insight into the SAM-mediated assembly of the mitochondrial TOM core complex. Science. (2021), 373(6561): 1377-81.
  3. Soto, C. and Pritzkow, S., Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat. Neurosci. (2018), 21(10): 1332-1340.