Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

7-POS Board 3 CONTEXTUALIZING STRUCTURAL INTERACTIONS OF THE FIP200 CLAW DOMAIN David Nyenhuis 1 ; William Rosencrans 2 ; Daniel Morris 1 ; Rebecca Bernstein 1 ; Richard Youle 2 ; Nico Tjandra 1 ; 1 National Institutes of Health, NHLBI, Bethesda, MD, USA 2 National Institutes of Health, NINDS, Bethesda, MD, USA Autophagy is a cellular process wherein a double-membrane is constructed to sequester cytoplasmic cargo, which is then routed to the lysosome for degradation. Autophagy can be either general, in response to metabolic changes and stress, or selective. The latter is mediated through an assortment of autophagic receptors to degrade pathogens, damaged mitochondria, and other specific cargo. Dysregulation of autophagy is associated with neurodegenerative disorders and cancer pathologies. Upstream formation of the core autophagic machinery involves the unc 51-like kinase 1 (ULK1) complex, which also contains focal adhesion kinase family interacting protein of 200 kDa (FIP200) and the autophagy related (ATG) proteins 13 and 101. FIP200 is a long (~1600 residue), dimeric scaffolding protein whose “C-shaped” N-terminal domain mediates ULK1 complex interactions. A long linker and extended coiled-coil domain then structurally isolate the N-terminus from the C-terminal CLAW domain, which recognizes FIP200-interacting region (FIR) motifs in autophagic receptors. The CLAW monomer comprises a six- stranded β -sheet, a single a-helix, and three elongated loops that form a “claw like” shape. Several structures have been solved for the FIP200 CLAW dimer in the apo state, with bound polyanions, and in complex with FIR containing peptides from several autophagic receptors. FIR binding impacts the dimer conformation, and is putatively modulated by motif phosphorylation, pointing to conformational regulation of FIP200-receptor interactions. We used solution and paramagnetic NMR to examine binding events and structural transitions of the CLAW domain in response to polyanions and the FIR motifs of autophagic receptors. Using both NMR and electron microscopy, we then found that these interactions are modulated by membrane environments, with ramifications for the specificity of the CLAW domain and potential temporal organization of FIP200 binding partners in the initial stages of autophagy.

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