Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease.

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.

Plant protein-lipid interfaces studied by molecular dynamics simulations.

The delineation of protein-lipid interfaces is essential for understanding the mechanisms of various membrane-associated processes crucial to plant development and growth, including signalling, trafficking, and membrane transport. Due to their highly dynamic nature, the precise characterization of lipid-protein interactions by experimental techniques is challenging. Molecular dynamics simulations provide a powerful computational alternative with a spatial-temporal resolution allowing the atomistic-level description. In this review, we aim to introduce plant scientists to molecular dynamics simulations. We describe different steps of performing molecular dynamics simulations and provide a broad survey of molecular dynamics studies investigating plant protein-lipid interfaces. Our aim is also to illustrate that combining molecular dynamics simulations with artificial intelligence-based protein structure determination opens up unprecedented possibilities for future investigations of dynamic plant protein-lipid interfaces.

Exploring lipid-protein interactions in plant membranes.

Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein-lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein-lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein-lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein-lipid interactions in plant cell biology.

Structural studies of a serum amyloid A octamer that is primed to scaffold lipid nanodiscs.

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.

Assessing the Martini 3 protein model: A review of its path and potential.

Coarse-grained (CG) protein models have become indispensable tools for studying many biological protein details, from conformational dynamics to the organization of protein macro-complexes, and even the interaction of proteins with other molecules. The Martini force field is one of the most widely used CG models for bio-molecular simulations, partly because of the enormous success of its protein model. With the recent release of a new and improved version of the Martini force field - Martini 3 - a new iteration of its protein model was also made available. The Martini 3 protein force field is an evolution of its Martini 2 counterpart, aimed at improving many of the shortcomings that had been previously identified. In this mini-review, we first provide a general overview of the model and then focus on the successful advances made in the short time since its release, many of which would not have been possible before. Furthermore, we discuss reported limitations, potential directions for model improvement and comment on what the likely future development and application avenues are.

Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Dimeric Tubulin Modifies Mechanical Properties of Lipid Bilayer, as Probed Using Gramicidin A Channel.

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.

Advances in Membrane Mimetic Systems for Manipulation and Analysis of Membrane Proteins: Detergents, Polymers, Lipids and Scaffolds.

Extracting membrane proteins from the hydrophobic environment of the biological membrane, in a physiologically relevant and stable state, suitable for downstream analysis remains a challenge. The traditional route to membrane protein extraction has been to use detergents and the last 15 years or so have seen a veritable explosion in the development of novel detergents with improved properties, making them more suitable for individual proteins and specific applications. There have also been significant advances in the development of encapsulation of membrane proteins in lipid based nanodiscs, either directly from the native membrane using polymers allowing effective capture of the protein and protein-associated membrane lipids, or via reconstitution of detergent extracted and purified protein into nanodiscs of defined lipid composition. All of these advances have been successfully applied to the study of membrane proteins via a range of techniques and there have been some spectacular membrane protein structures solved. In addition, the first detailed structural and biophysical analyses of membrane proteins retained within a biological membrane have been reported. Here we summarise and review the recent advances with respect to these new agents and systems for membrane protein extraction, reconstitution and analysis.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

PLAT domain protein 1 (PLAT1/PLAFP) binds to the Arabidopsis thaliana plasma membrane and inserts a lipid.

Robust agricultural yields depend on the plant's ability to fix carbon amid variable environmental conditions. Over seasonal and diurnal cycles, the plant must constantly adjust its metabolism according to available resources or external stressors. The metabolic changes that a plant undergoes in response to stress are well understood, but the long-distance signaling mechanisms that facilitate communication throughout the plant are less studied. The phloem is considered the predominant conduit for the bidirectional transport of these signals in the form of metabolites, nucleic acids, proteins, and lipids. Lipid trafficking through the phloem in particular attracted our attention due to its reliance on soluble lipid-binding proteins (LBP) that generate and solubilize otherwise membrane-associated lipids. The Phloem Lipid-Associated Family Protein (PLAFP) from Arabidopsis thaliana is generated in response to abiotic stress as is its lipid-ligand phosphatidic acid (PA). PLAFP is proposed to transport PA through the phloem in response to drought stress. To understand the interactions between PLAFP and PA, nearly 100 independent systems comprised of the protein and one PA, or a plasma membrane containing varying amounts of PA, were simulated using atomistic classical molecular dynamics methods. In these simulations, PLAFP is found to bind to plant plasma membrane models independent of the PA concentration. When bound to the membrane, PLAFP adopts a binding pose where W41 and R82 penetrate the membrane surface and anchor PLAFP. This triggers a separation of the two loop regions containing W41 and R82. Subsequent simulations indicate that PA insert into the ß-sandwich of PLAFP, driven by interactions with multiple amino acids besides the W41 and R82 identified during the insertion process. Fine-tuning the protein-membrane and protein-PA interface by mutating a selection of these amino acids may facilitate engineering plant signaling processes by modulating the binding response.

Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

SIMULTANEOUS NATIVE MASS SPECTROMETRY ANALYSIS OF SINGLE AND DOUBLE MUTANTS TO PROBE LIPID BINDING TO MEMBRANE PROTEINS.

Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that the W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into lipid binding to specific sites on membrane proteins.

Lipid composition modulates interactions of p7 viroporin during membrane insertion.

Viral proteins interact with lipid membranes during various stages in the viral life cycle to propagate infection. p7 is an ion channel forming protein of Hepatitis C virus (HCV) that participates in viral assembly. Studies show that it has close ties to lipid metabolism in the cell and anionic phosphatidylserine (PS) lipids are suggested to be key for its permeabilizing function, but the mechanism of its interaction with the lipid environment is largely unknown. To begin unraveling the molecular processes of the protein, we evaluated the impact of lipid environment on the binding and insertion mechanism of p7 prior to channel formation and viral assembly using molecular dynamics simulations. It is seen that p7 is sensitive to its lipid environment and results in different remodeling patterns in membranes. Helix 1 (H1) is especially important for peptide insertion, with deeper entry taking place when the membrane contains phosphatidylserine (PS). Helix 2 (H2) and the adjacent loop connecting to Helix 3 (H3) prompts recruitment of phosphatidylethanolamine (PE) lipids to the protein binding site in membrane models with lower surface charge. This work provides perspectives on the interplay between protein-lipid dynamics and membrane composition, and insights on membrane reorganization in mechanisms of disease.

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aß) peptides in the brain. The processing of amyloid precursor protein (APP) into Aß is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aß1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate.

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

Dissecting the contributions of membrane affinity and bivalency of the spider venom protein DkTx to its sustained mode of TRPV1 activation.

The spider venom protein, double-knot toxin (DkTx), partitions into the cellular membrane and binds bivalently to the pain-sensing ion channel, TRPV1, triggering long-lasting channel activation. In contrast, its monovalent single knots membrane partition poorly and invoke rapidly reversible TRPV1 activation. To discern the contributions of the bivalency and membrane affinity of DkTx to its sustained mode of action, here, we developed diverse toxin variants including those containing truncated linkers between individual knots, precluding bivalent binding. Additionally, by appending the single-knot domains to the Kv2.1 channel-targeting toxin, SGTx, we created monovalent double-knot proteins that demonstrated higher membrane affinity and more sustained TRPV1 activation than the single-knots. We also produced hyper-membrane affinity-possessing tetra-knot proteins, (DkTx)2 and DkTx-(SGTx)2, that demonstrated longer-lasting TRPV1 activation than DkTx, establishing the central role of the membrane affinity of DkTx in endowing it with its sustained TRPV1 activation properties. These results suggest that high membrane affinity-possessing TRPV1 agonists can potentially serve as long-acting analgesics.

Membrane interactions of apoptotic inhibitor Bcl-xL: What can be learned using fluorescence spectroscopy.

Permeabilization of the mitochondrial outer membrane-a point of no return in apoptotic regulation-is tightly controlled by proteins of the Bcl-2 family. Apoptotic inhibitor Bcl-xL is an important member of this family, responsible for blocking the permeabilization, and is also a promising target for anti-cancer drugs. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (i) a soluble folded conformation, (ii) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (iii) refolded membrane-inserted conformations, for which no structural data are available. In this review, we present the summary of the application of various methods of fluorescence spectroscopy for studying membrane interaction of Bcl-xL, and specifically the formation of the refolded inserted conformation. We discuss the application of environment-sensitive probes, Förster resonance energy transfer, fluorescence correlation spectroscopy, and fluorescent quenching for structural, thermodynamic, and functional characterization of protein-lipid interactions, which can benefit studies of other members of Bcl-2 (e.g., Bax, BAK, Bid). The conformational switching between various conformations of Bcl-xL depends on the presence of divalent cations, pH and lipid composition. This insertion-refolding transition also results in the release of the BH4 regulatory domain from the folded structure of Bcl-xL, which is relevant to the lipid-regulated conversion between canonical and non-canonical modes of apoptotic inhibition.

Cholesterol in Class C GPCRs: Role, Relevance, and Localization.

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.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale.

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. µs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.