TMEM16 scramblases thin the membrane to enable lipid scrambling

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. It was proposed that lipid headgroups move between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryoEM structure of the Ca2+-bound afTMEM16 scramblase in nanodiscs showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove’s intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not entail specific protein-lipid interactions with the extracellular vestibule. Further, we find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes despite an open pathway. Our results show how afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.

Mechanical and molecular activation lead to structurally analogous MscL states

The mechanosensitive channel of large conductance MscL gates in response to tension changes in the membrane to allow the exchange of molecules through its pore. Native ligands that bind and modulate MscL are unknown and trapping an activated state has been challenging. Disruption of lipid access to tension-sensitive transmembrane pockets by modification leads to a concerted structural and functional MscL response. However, it is unknown whether there is structural correlation between tension mediated and molecular activation in mechanosensitive channels. Here, we combine HDX mass spectrometry and ESEEM solvent accessibility measurements on MscL, coupled with molecular dynamics under bilayer tension, to investigate the structural changes associated with the two distinctively derived states. Membrane thinning is not sufficient to hydrate the MscL pore, when lipids are trapped in the pockets, under tensions capable to gate the native channel. Tension and molecule stabilised states present analogous MscL structures, suggesting a link between these two distinct activation mechanisms. These findings could hint at synergistic modes of regulation in mechanosensitive ion channels with implications for their multimodality.

The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.

Membrane thinning and lateral gating are consistent features of BamA across multiple species

In Gram-negative bacteria, the folding and insertion of β-barrel outer membrane proteins (OMPs) to the outer membrane are mediated by the β-barrel assembly machinery (BAM) complex. Two leading models of this process have been put forth: the hybrid barrel model, which claims that a lateral gate in BamA’s β-barrel can serve as a template for incoming OMPs, and the passive model, which claims that a thinned membrane near the lateral gate of BamA accelerates spontaneous OMP insertion. To examine the key elements of these two models, we have carried out 45.5 μs of equilibrium molecular dynamics simulations of BamA with and without POTRA domains from Escherichia coli, Salmonella enterica, Haemophilus ducreyi and Neisseria gonorrhoeae, together with BamA’s homolog, TamA from E. coli, in their native, species-specific outer membranes. In these equilibrium simulations, we consistently observe membrane thinning near the lateral gate for all proteins. We also see occasional spontaneous lateral gate opening and sliding of the β-strands at the gate interface for N. gonorrhoeae, indicating that the gate is dynamic. An additional 14 μs of free-energy calculations shows that the energy necessary to open the lateral gate in BamA/TamA varies by species, but is always lower than the Omp85 homolog, FhaC. Our combined results suggest OMP insertion utilizes aspects of both the hybrid barrel and passive models.

Roles of Membrane Domains in Integrin-Mediated Cell Adhesion

The composition and organization of the plasma membrane play important functional and regulatory roles in integrin signaling, which direct many physiological and pathological processes, such as development, wound healing, immunity, thrombosis, and cancer metastasis. Membranes are comprised of regions that are thick or thin owing to spontaneous partitioning of long-chain saturated lipids from short-chain polyunsaturated lipids into domains defined as ordered and liquid-disorder domains, respectively. Liquid-ordered domains are typically 100 nm in diameter and sometimes referred to as lipid rafts. We posit that integrin β senses membrane thickness and that mechanical force on the membrane regulates integrin activation through membrane thinning. This review examines what we know about the nature and mechanism of the interaction of integrins with the plasma membrane and its effects on regulating integrins and its binding partners.

Structural basis for membrane insertion by the human ER membrane protein complex

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit ER membrane protein complex (EMC) is a conserved co- and post-translational insertase at the endoplasmic reticulum. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.