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National Biomedical Center
for Advanced Electron Spin Resonance Technology

Our research is supported by a grant from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

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Structural Basis for Activation, Assembly and Membrane Binding of ESCRT-III Snf7 Filaments Elucidated by Pulse Dipolar ESR

The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament was unknown. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we showed that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity, giving rise to a membrane-sculpting filament. Then by using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrated that mutations on these protein interfaces abolish Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling. This study required the use of samples with very low concentrations of labeled membrane protein, less than 10 μM, demonstrating the possibilities offered by the high sensitivity of ACERT's PDS spectrometers.

Funding: P41GM105321, R01EB003150 (to JHF), T32GM007273 (to ST), R01GM094347 (to YM), R01GM098621 (to JCF); Amer. Cancer Soc. PF-12-026-01-DMC (to NJB); Cornell Univ. CU3704 (to SDE).

Publication: S. Tang, W.M. Henne, P.P. Borbat, N.J. Buchkovich, J.H. Freed, Y. Mao, J.C. Fromme, and S.D. Emr. eLife 4, e12548 (2015); PMCID: PMC4720517.

(A) PDS distance measurements on doubly spin-labeled Snf7 cysteine mutants revealed large conformation rearrangements upon transition from free-state closed conformation in solution to the open conformation in liposome-bound state. (B) PDS distance measurements on single spin-labeled mutants of Snf7 revealed that on the surface of liposome the protein assembles into large filaments with periodic structure.
S. Tang, W.M. Henne (Weill Institute of Cell and Molecular Biology, Cornell University, Ithaca; Department of Molecular Biology and Genetics, Cornell University, Ithaca)
P.P. Borbat (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY; ACERT)
N.J. Buchkovich (Weill Institute of Cell and Molecular Biology, Cornell University, Ithaca; Department of Molecular Biology and Genetics, Cornell University, Ithaca)
J.H. Freed (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY; ACERT)
Y. Mao, J.C. Fromme, and S.D. Emr (Weill Institute of Cell and Molecular Biology, Cornell University, Ithaca; Department of Molecular Biology and Genetics, Cornell University, Ithaca)
June, 2016