SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on protein stability and its conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in membrane bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366, and Arg371) that are located at the membrane-cytosol interface and consistently interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward-facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid polar heads. This results in a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt-bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease.

Serum amyloid A (SAA) is a highly conserved acute-phase protein that plays roles in activating multiple pro-inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high-density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein-protein interaction sites on SAA. While high-resolution structures for lipid-free, or apo-, forms of SAA have been reported, their relationship with the HDL-bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC-MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid-free and lipid-bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring-like structures reported for lipid-free ApoA-I. These SAA octamers represent a previously uncharacterized structure for lipid-free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA-I. The SAA-lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid-free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid-containing particles such as HDL. This study suggests a new model for SAA-lipid interactions and provides new insight into how SAA might stabilize protein-lipid nanodiscs or even replace ApoA-I as a scaffold for HDL particles during inflammation.

Using the gramicidin A channel as a molecular probe, we show that tubulin binding to planar lipid membranes changes the channel kinetics-seen as an increase in the lifetime of the channel dimer-and thus points towards modification of the membrane\'s mechanical properties. The effect is more pronounced in the presence of non-lamellar lipids in the lipid mixture used for membrane formation. To interpret these findings, we propose that tubulin binding redistributes the lateral pressure of lipid packing along the membrane depth, making it closer to the profile expected for lamellar lipids. This redistribution happens because tubulin perturbs the lipid headgroup spacing to reach the membrane\'s hydrophobic core via its amphiphilic α-helical domain. Specifically, it increases the forces of repulsion between the lipid headgroups and reduces such forces in the hydrophobic region. We suggest that the effect is reciprocal, meaning that alterations in lipid bilayer mechanics caused by membrane remodeling during cell proliferation in disease and development may also modulate tubulin membrane binding, thus exerting regulatory functions. One of those functions includes the regulation of protein-protein interactions at the membrane surface, as exemplified by VDAC complexation with tubulin.

Phenol-soluble modulins (PSMs) are key virulence factors of S. aureus, and they comprise the structural scaffold of biofilm as they self-assemble into functional amyloids. They have been shown to interact with cell membranes as they display toxicity towards human cells through cell lysis, with αPSM3 being the most cytotoxic. In addition to causing cell lysis in mammalian cells, PSMs have also been shown to interact with bacterial cell membranes through antimicrobial effects. Here, we present a study on the effects of lipid bilayers on the aggregation mechanism of αPSM using chemical kinetics to study the effects of lipid vesicles on the aggregation kinetics and using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) to investigate the corresponding secondary structure of the aggregates. We found that the effects of lipid bilayers on αPSM aggregation were not homogeneous between lipid type and αPSM peptides, although none of the lipids caused changes in the dominating aggregation mechanism. In the case of αPSM3, all types of lipids slowed down aggregation to a varying degree, with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) having the most pronounced effect. For αPSM1, lipids had opposite effects, where DOPC decelerated aggregation and lipopolysaccharide (LPS) accelerated the aggregation, while 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) had no effect. For αPSM4, both DOPG and LPS accelerated the aggregation, but only at high concentration, while DOPC showed no effect. None of the lipids was capable of inducing aggregation of αPSM2. Our data reveal a complex interaction pattern between PSMs peptides and lipid bilayers that causes changes in the aggregation kinetics by affecting different kinetic parameters along with only subtle changes in morphology.

G-protein coupled receptors (GPCRs), one of the largest superfamilies of cell-surface receptors, are heptahelical integral membrane proteins that play critical roles in virtually every organ system. G-protein-coupled receptors operate in membranes rich in cholesterol, with an imbalance in cholesterol level within the vicinity of GPCR transmembrane domains affecting the structure and/or function of many GPCRs, a phenomenon that has been linked to several diseases. These effects of cholesterol could result in indirect changes by altering the mechanical properties of the lipid environment or direct changes by binding to specific sites on the protein. There are a number of studies and reviews on how cholesterol modulates class A GPCRs; however, this area of study is yet to be explored for class C GPCRs, which are characterized by a large extracellular region and often form constitutive dimers. This review highlights specific sites of interaction, functions, and structural dynamics involved in the cholesterol recognition of the class C GPCRs. We summarize recent data from some typical family members to explain the effects of membrane cholesterol on the structural features and functions of class C GPCRs and speculate on their corresponding therapeutic potential.

Borate transporters are membrane transport proteins that regulate intracellular borate levels. In plants, borate is a micronutrient essential for growth but is toxic in excess, while in yeast, borate is unnecessary for growth and borate export confers tolerance. Borate transporters share structural homology with human bicarbonate transporters in the SLC4 family despite low sequence identity and differences in transported solutes. Here, we characterize the S. cerevisiae borate transporter Bor1p and examine whether key biochemical features of SLC4 transporters extend to borate transporters. We show that borate transporters and SLC4 transporters share multiple properties, including lipid-promoted dimerization, sensitivity to stilbene disulfonate-derived inhibitors, and a requirement for an acidic residue at the solute binding site. We also identify several amino acids critical for Bor1p function and show that disease-causing mutations in human SLC4A1 will eliminate in vivo function when their homologous mutations are introduced in Bor1p. Our data help elucidate mechanistic features of Bor1p and reveal significant functional properties shared between borate transporters and SLC4 transporters.

GPCRs receive signals from diverse messengers and activate G proteins that regulate downstream signaling effectors. Efficient signaling is achieved through the organization of these proteins in membranes. Thus, protein-lipid interactions play a critical role in bringing G proteins together in specific membrane microdomains with signaling partners. Significantly, the molecular basis underlying the membrane distribution of each G protein isoform, fundamental to fully understanding subsequent cell signaling, remains largely unclear. We used model membranes with lipid composition resembling different membrane microdomains, and monomeric, dimeric and trimeric Gi proteins with or without single and multiple mutations to investigate the structural bases of G protein-membrane interactions. We demonstrated that cationic amino acids in the N-terminal region of the Gαi1 and C-terminal region of the Gγ2 subunit, as well as their myristoyl, palmitoyl and geranylgeranyl moieties, define the differential G protein form interactions with membranes containing different lipid classes (PC, PS, PE, SM, Cho) and the various microdomains they may form (Lo, Ld, PC bilayer, charged, etc.). These new findings in part explain the molecular basis underlying amphitropic protein translocation to membranes and localization to different membrane microdomains and the role of these interactions in cell signal propagation, pathophysiology and therapies targeted to lipid membranes.

When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.

Over the past few years, the attention of the whole world has been riveted to the emergence of new dangerous strains of viruses, among which a special place is occupied by coronaviruses that have overcome the interspecies barrier in the past 20 years: SARS viruses (SARS), Middle East respiratory syndrome (MERS), as well as a new coronavirus infection (SARS-CoV-2), which caused the largest pandemic since the Spanish flu in 1918. Coronaviruses are members of a class of enveloped viruses that have a lipoprotein envelope. This class also includes such serious pathogens as human immunodeficiency virus (HIV), hepatitis, Ebola virus, influenza, etc. Despite significant differences in the clinical picture of the course of disease caused by enveloped viruses, they themselves have a number of characteristic features, which determine their commonality. Regardless of the way of penetration into the cell-by endocytosis or direct fusion with the cell membrane-enveloped viruses are characterized by the following stages of interaction with the target cell: binding to receptors on the cell surface, interaction of the surface glycoproteins of the virus with the membrane structures of the infected cell, fusion of the lipid envelope of the virion with plasma or endosomal membrane, destruction of the protein capsid and its dissociation from the viral nucleoprotein. Subsequently, within the infected cell, the newly synthesized viral proteins must self-assemble on various membrane structures to form a progeny virion. Thus, both the initial stages of viral infection and the assembly and release of new viral particles are associated with the activity of viral proteins in relation to the cell membrane and its organelles. This review is devoted to the analysis of physicochemical mechanisms of functioning of the main structural proteins of a number of enveloped viruses in order to identify possible strategies for the membrane activity of such proteins at various stages of viral infection of the cell.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.