Mpox is a zoonotic disease endemic in central and west Africa. However, since 2022, human-adapted mpox virus (MPXV) strains are causing large outbreaks spreading outside these regions, leading the World Health Organization to declare public health emergency twice. Tecovirimat, the most widely used drug to treat these infections, blocks viral egress through a poorly understood mechanism. Tecovirimat-resistant strains, all with mutations in the viral phospholipase F13, pose public health concerns. Herein, we report the structure of an F13 homodimer, both alone and in complex with tecovirimat. We demonstrate that tecovirimat acts as a molecular glue, inducing the dimerization of the phospholipase. F13 escape mutations in MPXV clinical isolates are at the dimer interface and prevent drug-induced dimerization in solution and cells. These findings, which decipher tecovirimat’s mode of action, will allow better monitoring of poxvirus outbreaks and pave the way for developing more potent and resilient therapeutics.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H⁺ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H⁺ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Lipid droplet (LD) function relies on proteins partitioning between the endoplasmic reticulum (ER) phospholipid bilayer and the LD monolayer membrane to control cellular adaptation to metabolic changes. It has been proposed that these hairpin proteins integrate into both membranes in a similar monotopic topology, enabling their passive lateral diffusion during LD emergence at the ER. Here, we combine biochemical solvent-accessibility assays, electron paramagnetic resonance spectroscopy and intra-molecular crosslinking experiments with molecular dynamics simulations, and determine distinct intramembrane positionings of the ER/LD protein UBXD8 in ER bilayer and LD monolayer membranes. UBXD8 is deeply inserted into the ER bilayer with a V-shaped topology and adopts an open-shallow conformation in the LD monolayer. Major structural rearrangements are required to enable ER-to-LD partitioning. Free energy calculations suggest that such structural transition is unlikely spontaneous, indicating that ER-to-LD protein partitioning relies on more complex mechanisms than anticipated and providing regulatory means for this trans-organelle protein trafficking.