114.2 - Molecular-scale structure of a high-curvature membrane

Adam Frost (UCSF), Frank Moss III (UCSF, SLAC@Stanford), James Lincoff (UCSF), Maxwell Tucker (UCSF), Arshad Mohammed (UCSF), Michael Grabe (UCSF, UCSF)

Cells utilize molecular machines to form and remodel their membrane-defined compartments’ compositions, shapes, and connections. The regulated activity of these membrane remodeling machines drives processes like vesicular traffic and organelle homeostasis. Although molecular patterning within membranes is essential to cellular life, characterizing the composition and structure of realistic biological membranes on the molecular length scale remains a challenge, particularly during membrane shape transformations. Here, we employed an ESCRT-III protein coating model system to investigate how membrane-binding proteins bind to and alter the structural patterns within lipid bilayers. We observe leaflet-level and localized lipid structures within a constricted and thinned membrane nanotube. To map the fine structure of these membranes, we compared simulated bilayer nanotubes with experimental cryo-EM reconstructions of native membranes and membranes containing halogenated lipid analogs. Halogenated lipids scatter electrons more strongly, and analysis of their surplus scattering enabled us to estimate the concentrations of lipids within each leaflet and to estimate lipid shape and sorting changes induced by high curvature and lipid-protein interactions. Specifically, we found that cholesterol enriched within the inner leaflet due to its spontaneous curvature, while acidic lipids enriched in the outer leaflet due to electrostatic interactions with the protein coat. The docosahexaenoyl (DHA) polyunsaturated chain-containing lipid SDPC enriched strongly at membrane-protein contact sites. Simulations and imaging of brominated SDPC showed how a pair of phenylalanine residues opens a hydrophobic defect in the outer leaflet and how DHA tails stabilize the defect and “snorkel” up to the membrane surface to interact with these side chains. This highly curved nanotube differs markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion, and other structural asymmetries with implications for our understanding of membrane mechanics.

This work was supported by an NIH postdoctoral fellowship to J.L. (4T32HL007731-28), NIH R01 GM117593 (M.G.), NIH P50 GM082545 (A.F.), NIH 1DP2-GM110772 (A.F.), and
hardware for simulations was provided by NIH R01GM089740 (M.G.). Simulations were also
carried out on the UCSF Wynton Cluster made possible through NIH 1S10OD021596 and
1S10OD020054-011. Microscopes are supported by NIH 1S10OD021741-01 and NIH
S10OD026881-01. A.F. is further supported by a Faculty Scholar grant from the HHMI and is a
Chan Zuckerberg Biohub investigator. F.M. is supported by a postdoctoral fellowship from The
Jane Coffin Childs Memorial Fund for Medical Research. Structural biology applications used in
this project were compiled and configured by SBGrid. The GPUs used for this research were
donated by the NVIDIA Corporation. Part of this work was performed at the Stanford Nano Shared
Facilities (SNSF), supported by the National Science Foundation (ECCS-1542152). Mass
spectrometry was performed at the Vincent Coates Foundation Mass Spectrometry Laboratory,
Stanford University Mass Spectrometry. This work was supported in part by NIH P30 CA124435
utilizing the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource.