Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Wednesday Speaker Abstracts

CRYO-EM STRUCTURES REVEAL PROGRESSIVE MEMBRANE CONSTRICTION BY THE ESCRT-III PROTEINS IST1 AND CHMP1B Henry C Nguyen 1 ; Nathaniel Talledge 2 ; John McCullough 2 ; Wesley I Sundquist 2 ; Adam Frost 1 ; 1 University of California, San Francisco, Biochemistry and Biophysics, San Francisco, California, USA 2 University of Utah, Biochemistry, Salt Lake City, Utah, USA The Endosomal Sorting Complexes Required for Transport (ESCRT) mediate critical membrane remodeling events throughout the mammalian cell cycle, including, but not limited to, HIV budding, cytokinetic abscission, and sealing of the nuclear envelope. ESCRT-III proteins polymerize into membrane-binding filaments to catalyze these reactions, but the structures and functions of these assemblies remain poorly understood. Our collaborative team recently determined the first atomic-resolution structure of an ESCRT-III filament – a hetero-polymer consisting of IST1 and CHMP1B. Our previous structure demonstrated how one of these subunits, CHMP1B, transitions from a “closed” to an “open” state to form an interlocked and domain-swapped filament. Moreover, we and others have shown that the IST1-CHMP1B copolymer participates in non-canonical, positive-curvature membrane fission pathways. Very recent work on other ESCRT-III proteins indicated that the mechanisms of opening and assembly we reported are conserved, but also raised questions regarding membrane binding and remodeling activities. To address these gaps in our understanding, we have determined the high- resolution structure of a membrane-bound IST1-CHMP1B assembly by cryo electron microscopy (cryoEM). We find that CHMP1B induces a high degree of curvature alone, and that deposition of the IST1 strand further constricts the membrane by more than 2-fold – almost to the fission point. Notably, the distance between outer leaflet lipid headgroups is ~10 nm and the distance between inner leaflet lipid headgroups is reduced to only ~4 nm. Conserved residues along helix a1 of CHMP1B serve as the major membrane binding surface and exploit both electrostatic as well as hydrophobic interactions with the convex leaflet of the membrane tubule. Our atomic-resolution cryoEM study reveals the structural mechanisms governing ESCRT-III assembly, membrane-binding, and positive-curvature membrane deforming activities.

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