TMEM16A is a Ca2+-activated anion channel that provides direct electrical feedback to the plasma membrane in response to intracellular Ca2+. Its conductive state remains unresolved, leaving questions about gating, Cl- permeation, and modulation by Ca2+, depolarization, and lipids. To investigate the open state, we performed molecular dynamics simulations of TMEM16A bound to the putative open-state blocker 1PBC. After inhibitor removal, the putative, pore-lining helix TM4 developed kinks at two sites: an upper site that opens the pore for Cl- permeation and a deeper site causing constriction. A conserved hydrophobic network between TM3 and TM4 persisted in most open structures but separated during extreme dilation, allowing lipids to transiently block the pore. Patch-clamp recordings indicated that the intact network promotes activation. Further simulations yielded >60 Cl- permeation events and a single-channel conductance that matched experiment. Additional electrostatic and kinetic modeling indicated that TMEM16A's transition from outward-rectification to ohmic conductance with increasing Ca2+ results from a weak voltage dependence of binding of Ca2+ ions, which acts cooperatively to open the pore.

The SARS CoV 2 accessory protein Orf9b is in a complex monomer-dimer equilibrium that influences its interactions with the host mitochondrial receptor Tom70. This interaction is critical for viral suppression of a Type-1 interferon response during infection. Modulating this equilibrium with a small molecule, either by stabilizing the Orf9b dimer or blocking its interaction with Tom70, represents a promising strategy for restoring interferon signaling and the antiviral response. To build tool molecules that could test this concept, we performed two screens: a crystallographic fragment screen against the Orf9b homodimer and a high-throughput fluorescence polarization screen for competitors of an Orf9b-derived peptide binding to Tom70. Fragment screening revealed two binding sites with potential to be developed into an inhibitor: one located at the peripheral dimer interface and the other just outside the lipid-binding channel that defines the central dimer interface. Functionalization of the fragments outside of the lipid-binding channel with hydrophobic moieties stabilized the Orf9b dimer thereby indirectly inhibiting association with Tom70. In parallel, the high throughput screen for competitive inhibitors of the Tom70:Orf9b interaction discovered a separate series of molecules. These molecules display dynamic structure activity relationship (SAR) and could be improved in the future to modulate the interaction between Tom70 and potentially a wide range of substrates. Collectively, these results demonstrate the feasibility of two distinct strategies to manipulate the Orf9b-Tom70 equilibrium, which is critical to the host response to SARS CoV 2 infection.

The organic anion transporting polypeptide (OATP)-1B1 and -1B3 are liver-specific transporters that govern the uptake of numerous endogenous molecules and drugs before their metabolism and excretion by the hepatocytes. Structurally, these two transporters are members of the major facilitator superfamily, operating by the alternating access mechanism that facilitates the movement of solutes between extracellular and intracellular compartments. Given their dynamic nature, salt bridges often modulate the conformations of transporters and participate in the orchestration of conformational changes. In this study, we identified and characterized a network of salt bridges within the internal cavities of OATP1B1 and OATP1B3 by cell-based uptake assays, uptake kinetics, and molecular dynamics simulations. These experiments revealed that a salt bridge network centered around E185 is crucial for uptake activities in these two proteins, as it stabilizes the inward cavity of the proteins and bridges the N- and C- bundles of the protein. Interestingly, this salt bridge network changes as a function of conformation. Furthermore, the residues studied do not participate in ligand coordination in the published structures nor in our simulations. These findings advance our understanding of the elaborate network of ionic interactions that govern the structure and dynamics of OATP1B1, OATP1B3, and other MFS transporters.

Cryo-electron microscopy (cryo-EM) is a powerful tool for atomic- and molecular-resolution structure determination, whereas molecular dynamics (MD) simulations are similarly powerful tools for predicting molecular trajectories. Given the challenges in estimating biomolecule dynamics with cryo-EM alone, MD simulations are employed to forecast molecular motions and to interpret cryo-EM reconstructions. Few methods, however, can evaluate MD predictions directly. Here, we use multislice wave propagation to project sampled snapshots of MD trajectories, either coarse grained or all atom, into simulated cryo-EM 3D reconstructions. We compared simulated and experimental images of low- and high-curvature membranes to show that MD simulations qualitatively reflect the fluidity and thus the contrast of biological membranes observed by cryo-EM. MD simulations also correctly predicted bilayer dimensions for single-component flat bilayers observed in cryo-EM images. However, Martini3 coarse-grained MD simulations failed to predict changes in membrane thickness induced by high curvature and with heterogeneous lipid compositions. We pinpointed the misbehavior of polyunsaturated lipid tails and cholesterol in Martini3 simulations as the main error sources contributing to inaccurate bilayer thicknesses. Our comparisons also explain membrane structure discrepancies between cryo-EM and small angle x-ray scattering. Further testing of MD predictions by direct comparisons between simulated and experimental cryo-EM images should lead to the development of more accurate MD force fields.

Many therapeutically relevant membrane proteins possess druggable sites at the lipid-protein interface, but principles guiding ligand design for these sites are not well-defined. CFTR potentiators, a clinically validated drug class for cystic fibrosis, offer a compelling model for exploring membrane-targeted design principles, as their efficacy depends on sustained intramembrane binding to prolong channel opening and chloride conductance. Here, we systematically modified the lipophilic substituent of the CFTR potentiator ABBV-974 and identified an analog that confers markedly increased functional residence time, as measured by delayed current decay after compound washout in patch-clamp assays. Kinetic analysis incorporating the physicochemical properties of the lipophilic substituents suggests that this increased kinetic stability may arise from an increased residence time in the cell membrane, consistent with qualitative results of molecular dynamics simulations. These results establish a structure-function link between membrane-facing ligand modifications and functional target engagement and potentially offer generalizable strategies for designing probe molecules and drugs that stably engage lipid-exposed binding sites on membrane proteins.

Imbalances in lipid storage and secretion lead to hepatic steatosis, the accumulation of lipid droplets in hepatocytes 1,2 . Our understanding of the mechanisms that govern the channelling of neutral lipids in hepatocytes towards cytosolic lipid droplets or secreted lipoproteins remains incomplete 3,4 . Here we performed a series of CRISPR–Cas9 screens under different metabolic states that led to the identification of CLCC1 as a critical regulator of neutral lipid storage and secretion in hepatocytes. Loss of CLCC1 resulted in the buildup of large lipid droplets in hepatoma cells and Clcc1 knockout in mice caused liver steatosis. Lipid droplets were present in the lumen of the endoplasmic reticulum of the Clcc1 -knockout hepatocytes and exhibited properties of lipoproteins, indicating a profound shift in neutral lipid flux. The loss of CLCC1 also led to the accumulation of nuclear membrane herniations accompanied by a reduction in nuclear pores. Remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1 and Brr6, factors that promote nuclear envelope fusion during nuclear pore complex assembly. Molecular dynamics simulations and mutagenesis studies support a model in which CLCC1 mediates membrane bending and fusion. We propose that CLCC1 mediates membrane fusion to promote hepatic neutral lipid flux and nuclear pore complex assembly.

Biological membranes contain a diverse set of membrane proteins surrounded by many different lipids, and the lateral organization and function of these molecules are closely intertwined. Here, we use coarse-grained molecular dynamics (MD) simulations to explore how hydrophobic mismatch between the length of transmembrane (TM) proteins and the thickness of the surrounding lipid membrane impacts the spatial distribution of the lipids. We constructed idealized cylindrically symmetric proteins, inspired by the “Mattress Model” developed in the 1980’s, and simulated these model proteins in different lipid compositions. We found that unsaturated lipids were attracted to short TM proteins that thinned the membrane, while fully saturated lipids were attracted to long TM proteins that induced membrane extension. A simple mechanical description of the membrane deformation energy coupled to a lipid mixing model accurately predicted the enrichment/depletion, which was up to 33% in some cases. Overall, we find that the main driver behind unsaturation-based sorting is the effective lipid length that conforms to match the hydrophobic thickness of the TM protein. Additionally, our simulations highlight that lipid sorting behavior is sensitive to protein tilt and protein surface roughness. By teasing out the fundamental physical principles in these simple models, our results provide a foundational understanding of how proteins and lipids form complex and transient assemblies, which we believe will be important for interpreting lipid-protein interactions for a host of membrane proteins that regulate cellular membranes and cell function.

Open Reading Frame 9b (Orf9b), an accessory protein of SARS-CoV and –2, is involved in innate immune suppression through its binding to the mitochondrial receptor Translocase of Outer Membrane 70 (Tom70). Previous structural studies of Orf9b in isolation revealed a β-sheet-rich homodimer; however, structures of Orf9b in complex with Tom70 revealed a monomeric helical fold. Here, we developed a biophysical model that quantifies how Orf9b switches between these conformations and binds to Tom70, a requirement for suppressing the type 1 interferon response. We used this model to characterize the effect of lipid binding and mutations in variants of concern to the Orf9b:Tom70 equilibrium. We found that the binding of a lipid to the Orf9b homodimer biases the Orf9b monomer:dimer equilibrium towards the dimer by reducing the dimer dissociation rate ~100 fold. We also found that mutations in variants of concern can alter different microscopic rate constants without significantly affecting binding to Tom70. Together, our results highlight how perturbations to different steps in these coupled equilibria can affect the apparent affinity of Orf9b to Tom70, with potential downstream implications for interferon signaling in coronavirus infection.

The asymmetric resting distribution of the three major phospholipid classes on the mammalian plasma membrane, with phosphatidylserine and phosphatidylethanolamine mostly on the inner leaflet and phosphatidylcholine mostly on the outer leaflet, is maintained by ATP-dependent flippases and floppases that exhibit headgroup selectivity. Upon signaling cues, this asymmetry can be dissipated by various phospholipid scramblases, allowing cells to respond to stimuli and adapt to different physiological contexts. The prevailing view in the field is that phospholipid scramblases on the plasma membrane act without headgroup preference. Here, we report contrary experimental evidence based on a phospholipid scrambling assay that quantifies the fluorescence polarization (FP) of nitrobenzoxadiazole (NBD)-labeled phospholipids for kinetic monitoring of phospholipid scrambling on the plasma membrane of living cells. Our experiments reveal that the plasma membrane-residing calcium-activated phospholipid scramblase TMEM16F preferentially acts on phosphatidylserine and phosphatidylcholine over phosphatidylethanolamine.

A hallmark of neurodegenerative diseases like Alzheimer's Disease (AD) and chronic traumatic encephalopathy (CTE) is the presence of toxic protein aggregates in neurons. In AD and CTE specifically, the protein tau forms insoluble fibrils that are hundreds of nanometers in length. Intriguingly, recent experimental structures suggest that tau ligands like the disaggregator EGCG and positron emission tomography (PET) tracers like GTP-1 and MK-6240 bind to tau fibrils in long stacks reflecting the symmetry of the protein across many binding sites. In these stacks, each ligand makes more contact with its symmetry mates than it does with the protein. To interpret the binding of these molecules and new ligands, we must understand the effects of the cooperativity between sites and the entropy coming from the number of sites. Here, we investigate a nearest-neighbors model of cooperativity and use statistical mechanics to derive binding isotherms for saturation and competition experiments. This model allows us to relate measured E C 50 and I C 50 values to the intrinsic binding affinity to a single site and to cooperativity across sites in ways resembling the Cheng-Prusoff Equation. Depending on the degree of cooperativity between molecular species, this model permits solutions that lack the steep binding curves expected from cooperative systems and even solutions resembling 2-site systems. We finally consider conditions for a fibril's detection in a PET scan and practical matters of fitting this model's parameters to data.