Conformation Of Membrane Proteins Research Articles

Efficiently determining membrane-bound conformations of peripheral membrane proteins using replica exchange with hybrid tempering

Efficiently determining membrane-bound conformations of peripheral membrane proteins using replica exchange with hybrid tempering

The application of nanodiscs in membrane protein drug discovery & development and drug delivery.

The phospholipid bilayer nanodiscs (LNDs), as a rapidly-developing tool in recent years, provide a natural bio-memebrane environment to maintain the native conformation and functions of membrane proteins as well as a versatile delivery vehicle for a variety of hydrophobic and hydrophilic drugs. We have seen unprecedented advantages of phospholipid bilayer nanodiscs in membrane protein structure characterization, biochemical and physiological studies of membrane proteins, membrane environment studies, drug discovery & development, and drug delivery. Many previous reviews have been mainly focused on the advantages of nanodiscs in membrane protein researches, but few have touched upon the importance and potential application of nanodiscs in pharmaceutical industries. This review will provide general description of the structural characteristics, advantages, classification, and applications of phospholipid nanodiscs, with particular focus on nanodisc-enabled membrane protein drug discovery & development as well as drug delivery.

A new preparation method of covalent annular nanodiscs based on MTGase

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.

Intrinsic Lipid Curvature and Bilayer Elasticity as Regulators of Channel Function: A Comparative Single-Molecule Study.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the HII lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature.

Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR

Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio). The resulting linewidths are about 30% sharper due to the higher orientational order parameter likely arising from the stabilizing electrostatic repulsion between the discs. Moreover, highly aligned, detergent-free macrodiscs can be formed with a longer-chain lipid, DPPC. Interestingly, the spectra of Pf1 in the two lipid mimetics are almost indistinguishable, which would mean that the overall transmembrane helix tilt might be governed not only by the hydrophobic matching but also possibly by the interactions of the flanking lysine and arginine residues at the membrane interface.

Effect of allyl isothiocyanate on the growth and virulence of Clostridium perfringens and its application on cooked pork

The objective of this study is to explore the antibacterial action modes and virulence-inhibitory effects of allyl isothiocyanate (AITC) against Clostridium perfringens (C. perfringens). The minimum inhibitory concentration (MIC) of AITC against vegetative cells of Cp 13124 was 0.1 μL/mL, and the time-kill kinetics analysis revealed that AITC could significantly suppress the growth of Cp 13124. According to the results from scanning electron microscopy (SEM), fluorescence microscopy, and UV absorbance substance detection, the cell membrane of Cp 13124 was damaged upon AITC treatment, causing a loss of integrity and the release of intracellular substances. Meanwhile, the fluorescence quenching experiment indicated the interaction of AIT-C with membrane proteins, which caused changes in the conformation of membrane proteins. Measurement of reactive oxygen species (ROS) and flow cytometry analysis demonstrated that AITC could induce apoptosis through oxidative stress. The formation of Cp 13124 biofilms was inhibited by AITC using the crystalline violet method, which was possibly related to the inhibition of sliding motility. Finally, low concentrations of AITC could be used as an antibacterial agent to inhibit the outgrowth of Cp 13124 in cooked pork, suggesting that AITC is a promising candidate for novel preservatives in the meat business.

Conformation of influenza AM2 membrane protein in nanodiscs and liposomes

The influenza A M2 protein (AM2) is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle including viral assembly and budding. An atomic-level conformational understanding of this key player in the influenza life cycle could inform new antiviral strategies. For conformational studies of complex systems like the AM2 membrane protein, a multipronged approach using different biophysical methods and different model membranes is a powerful way to incorporate complementary data and achieve a fuller, more robust understanding of the system. However, one must be aware of how the sample composition required for a particular method impacts the data collected and how conclusions are drawn. In that spirit, we systematically compared the properties of AM2 in two different model membranes: nanodiscs and liposomes. Electron paramagnetic spectroscopy of spin-labeled AM2 showed that the conformation and dynamics were strikingly similar in both AM2-nanodiscs and AM2-liposomes consistent with similar conformations in both model membranes. Analysis of spin labeled lipids embedded in both model membranes revealed that the bilayer in AM2-liposomes was more fluid and permeable to oxygen than AM2-nanodiscs with the same lipid composition. Once the difference in the partitioning of the paramagnetic oxygen relaxation agent was taken into account, the membrane topology of AM2 appeared to be the same in both liposomes and nanodiscs. Finally, functionally relevant AM2 conformational shifts previously seen in liposomes due to the addition of cholesterol were also observed in nanodiscs.

Mechanism of membrane damage to Shigella sonnei by linalool from plant essential oils: A driver of oxidative stress

This work explored the antibacterial mechanism of linalool on Shigella sonnei (S. sonnei) based on membrane damage driven by oxidative stress. The results revealed that linalool had excellent inhibition towards S. sonnei under a concentration of 1.5 mL/L. Furthermore, cell structure was disrupted with massive leakage of K+ and Mg2+ with linalool according to scanning electron microscopy, zeta potential and probes staining of fluorescein diacetate and Rhodamine 123 combined with fluorescence spectroscopy. Changes in membrane protein conformation were also monitored as a result of fluorescence spectroscopy when exposed to linalool. In addition, oxidative stress was appeared after exposure to linalool due to the increase of malondialdehyde, hydrogen peroxide, superoxide anion and oxidized glutathione, and the decrease of reduced glutathione. The activity of defense enzyme including superoxide dismutase, catalase and peroxidase, and antioxidant enzyme including glutathione reductase and glutathione S-transferase was also inhibited. In all, oxidative stress-driven membrane damage was considered as the main mechanism by which linalool inhibited S. sonnei. Our findings provide theoretical support for linalool as a promising natural antibacterial agent against S. sonnei contamination.

Effect of polyunsaturated free fatty acids on the membrane fusion mechanism.

Membrane fusion is one of the important processes for the survival of eukaryotic cells and the entry of enveloped viruses into the host cells. Lipid composition plays a crucial role by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. The diversity of the lipid acyl chain in its length and degree of unsaturation originates from the variation in free fatty acids (FFAs). We have studied the effect of linoleic (LA) and alpha-linolenic (ALA) acids on the depth-dependent organization, dynamics, and fusion of DOPC/DOPE (70/30 mol%) membranes utilizing steady-state and time-resolved fluorescence spectroscopic methods. Our results suggest that membranes with 5 mol% LA stabilize the stalk-intermediate and promote lipid mixing at the early stage of the process, i.e., the fusion follows the classical stalk model. Conversely, the extents of lipid and content mixing at the stalk intermediate are similar in the presence of 5 mol% of ALA, indicating the fusion mechanism as a nonclassical one like in the DOPC/DOPE (70/30 mol%) membranes. Our results provide an in-depth insight into the effect of the increasing degree of fatty acid tail unsaturation on membrane organization and dynamics and their impact on the membrane fusion mechanism.

Reconciling membrane protein simulations with experimental DEER spectroscopy data.

Spectroscopy experiments are crucial to study membrane proteins for which traditional structure determination methods still prove challenging. Double electron-electron resonance (DEER) spectroscopy experiments provide protein residue-pair distance distributions that are indicative of their conformational heterogeneity. Atomistic molecular dynamics (MD) simulations are another tool that have been proven to be vital to study the structural dynamics of membrane proteins such as to identify inward-open, occluded, and outward-open conformations of transporter membrane proteins, among other partially open or closed states of the protein. Yet, studies have reported that there is no direct consensus between the distributional data from DEER experiments and MD simulations, which has challenged validation of structures obtained from long-timescale simulations and using simulations to design experiments. Current coping strategies for comparisons rely on heuristics, such as mapping the nearest matching peaks between two ensembles or biased simulations. Here we examine the differences in residue-pair distance distributions arising due to the choice of membranes around the protein and covalent modification of a pair of residues to nitroxide spin labels in DEER experiments. Through comparing MD simulations of two proteins, PepTSo and LeuT-both of which have been characterized using DEER experiments previously-we show that the proteins' dynamics are similar despite the choice of the detergent micelle as a membrane mimetic in DEER experiments. On the other hand, covalently modified residues show slight local differences in their dynamics and a huge divergence when the oxygen atom pair distances between spin labeled residues are measured rather than protein backbone distances. Given the computational expense associated with pairwise MTSSL labeled MD simulations, we examine the use of biased simulations to explore the conformational dynamics of the spin labels only to reveal that such simulations alter the underlying protein dynamics. Our study identifies the main cause for the mismatch between DEER experiments and MD simulations and will accelerate the development of potential mitigation strategies to improve the match.

Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction.

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein-protein and protein-lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor-ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

A comparative study of interfacial environments in lipid nanodiscs and vesicles

Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for in vitro studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ∼10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ∼100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.

Antibacterial Activity and Mechanism of Polygonum orientale L. Essential Oil against Pectobacterium carotovorum subsp. carotovorum.

Infected by Pectobacterium carotovorum subsp. carotovorum (Pcc), the quality of Chinese cabbage could severely decline. Using chemical bactericides to control Pcc could cause food safety problems. Thus, we investigated the optimum extraction conditions, antibacterial activity, chemical compounds and antibacterial mechanism of Polygonum orientale L. essential oil (POEO) against Pcc in order to search a new way to control Pcc. The optimum extraction conditions of POEO (soaking time 2.6 h, extraction time 7.7 h and ratio of liquid to solid 10.3 mL/g) were optimized by response surface methodology. The minimum inhibitory concentration (MIC) of POEO against Pcc was 0.625 mg/mL. The control efficiency of protective activity of POEO against Pcc was 74.67~92.67%, and its curative activity was 76.00~93.00%. Then, 29 compounds were obtained by GC-MS; the prime compounds of POEO were phytol, phytone, n-pentacosane, 1-octen-3-ol and β-ionone. It was verified that, compared with control samples, POEO destroyed cell morphology. It increased surface potential, increased hydrophobicity, damaged cell walls, destroyed the integrity and permeability of cell membrane, reduced membrane potential (MP), and changed membrane protein conformation. It inhibited the activities of pyruvate kinase (PK), succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase). Briefly, the results of this study demonstrate that POEO showed effective inhibitory activity against Pcc, thus POEO could have potential application in controlling Pcc.

Measuring Membrane Penetration Depths and Conformational Changes in Membrane Peptides and Proteins.

The structural organization and dynamic nature of the biomembrane components are important determinants for numerous cellular functions. Particularly, membrane proteins are critically important for various physiological functions and are important drug targets. The mechanistic insights on the complex functionality of membrane lipids and proteins can be elucidated by understanding the interplay between structure and dynamics. In this regard, membrane penetration depth represents an important parameter to obtain the precise depth of membrane-embedded molecules that often define the conformation and topology of membrane probes and proteins. In this review, we discuss about the widely used fluorescence quenching-based methods (parallax method, distribution analysis, and dual-quencher analysis) to accurately determine the membrane penetration depths of fluorescent probes that are either membrane-embedded or attached to lipids and proteins. Further, we also discuss a relatively novel fluorescence quenching method that utilizes tryptophan residue as the quencher, namely the tryptophan-induced quenching, which is sensitive to monitor small-scale conformational changes (short distances of < 15Å) and useful in mapping distances in proteins. We have provided numerous examples for the benefit of readers to appreciate the importance and applicability of these simple yet powerful methods to study membrane proteins.

Emulating Membrane Protein Environments─How Much Lipid Is Required for a Native Structure: Influenza S31N M2.

This report investigates the homotetrameric membrane protein structure of the S31N M2 protein from Influenza A virus in the presence of a high molar ratio of lipid. The structured regions of this protein include a single transmembrane helix and an amphipathic helix. Two structures of the S31N M2 conductance domain from Influenza A virus have been deposited in the Protein Data Bank (PDB). These structures present different symmetries about the channel main axis. We present new magic angle spinning and oriented sample solid-state NMR spectroscopic data for S31N M2 in liquid crystalline lipid bilayers using protein tetramer:lipid molar ratios ranging from 1:120 to 1:240. The data is consistent with an essentially 4-fold-symmetric structure very similar to the M2 WT structure that also has a single conformation for the four monomers, except at the His37 and Trp41 functional sites when characterized in samples with a high molar ratio of lipid. While detergent solubilization is well recognized today as a nonideal environment for small membrane proteins, here we discuss the influence of a high lipid to protein ratio for samples of the S31N M2 protein to stabilize an essentially 4-fold-symmetric conformation of the M2 membrane protein. While it is generally accepted that the chemical and physical properties of the native environment of membrane proteins needs to be reproduced judiciously to achieve the native protein structure, here we show that not only the character of the emulated membrane environment is important but also the abundance of the environment is important for achieving the native structure. This is a critical finding as a membrane protein spectroscopist's goal is always to generate a sample with the highest possible protein sensitivity while obtaining spectra of the native-like structure.

Effect of Phosphatidylethanolamine and Oleic Acid on Membrane Fusion: Phosphatidylethanolamine Circumvents the Classical Stalk Model.

Membrane fusion is one of the most important processes for the survival of eukaryotic cells and entry of enveloped viruses to the host cells. Lipid composition plays a crucial role in the process by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. Phosphatidylethanolamine (PE), a lipid molecule with intrinsic negative curvature, promotes membrane fusion by stabilizing the non-lamellar intermediate structures in the fusion process. Conversely, oleic acid (OA), with intrinsic positive curvature, inhibits membrane fusion. The current study aimed to investigate polyethylene glycol-mediated lipid mixing, content mixing, content leakage, and depth-dependent membrane organization and dynamics, using arrays of steady-state and time-resolved fluorescence techniques, to determine the causative role of PE and OA in membrane fusion. The results demonstrated that the presence of 30 mol % PE in the membrane promotes membrane fusion through a mechanism that circumvents the classical stalk model. On the contrary, membranes containing OA showed reduced rate and extent of fusion, despite following the same mechanism. Collectively, our findings in terms of membrane organization and dynamics indicated a plausible role of PE and OA in membrane fusion.

Exposure to 60 Hz magnetic field can affect membrane proteins and membrane potential in human cancer cells

ABSTRACT The experimental data support the hypothesis that extremely low frequency magnetic field (ELF-MF) can affect cell membranes. Since our previous studies suggested that MF changes the permeability of cell membrane, in this study we focused on the cell membrane and investigated the effect of 60 Hz, 50 mT MF on the membrane potential and membrane proteins. The membrane potentials of three cultured human cancer cell lines, A549, MES-SA, and MES-SA/Dx5, were increased by exposure to ELF-MF. When exposed to MF and an anticancer drug, changes in the membrane potentials were detected in A549 and MES-SA cells, but not in the multi drug-resistant cells, MES-SA/Dx5. We examined whether MF has an influence on the membrane proteins extracted from cultured A549 cells, using DiBAC4(3) dye enhanced fluorescence binding to a hydrophobic site. The increase in fluorescence observed following MF exposure for 10 min indicated that the structure of the hydrophobic site on the membrane proteins changed and became more likely to bind the probe dye. A decrease in fluorescence was detected following exposure to MF for 240 min. These results indicated that 60 Hz, 50 mT MF causes changes in the membrane potential of cultured cancer cells and the conformation of membrane proteins extracted from cultured cancer cells, and has different effects depending on the exposure time.

Solid-State NMR of Membrane Proteins in Lipid Bilayers: To Spin or Not To Spin?

Membrane proteins mediate a plethora of cellular functions and represent important targets for drug development. Unlike soluble proteins, membrane proteins require native-like environments to fold correctly and be active. Therefore, modern structural biology techniques have aimed to determine the structure and dynamics of these membrane proteins at physiological temperature and in liquid crystalline lipid bilayers. With the flourishing of new NMR methodologies and improvements in sample preparations, magic angle spinning (MAS) and oriented sample solid-state NMR (OS-ssNMR) spectroscopy of membrane proteins is experiencing a new renaissance. Born as antagonistic approaches, these techniques nowadays offer complementary information on the structural topology and dynamics of membrane proteins reconstituted in lipid membranes. By spinning biosolid samples at the magic angle (θ = 54.7°), MAS NMR experiments remove the intrinsic anisotropy of the NMR interactions, increasing spectral resolution. Internuclear spin interactions (spin exchange) are reintroduced by RF pulses, providing distances and torsion angles to determine secondary, tertiary, and quaternary structures of membrane proteins. OS-ssNMR, on the other hand, directly detects anisotropic NMR parameters such as dipolar couplings (DC) and anisotropic chemical shifts (CS), providing orientational constraints to determine the architecture (i.e., topology) of membrane proteins relative to the lipid membrane. Defining the orientation of membrane proteins and their interactions with lipid membranes is of paramount importance since lipid-protein interactions can shape membrane protein conformations and ultimately define their functional states.In this Account, we report selected studies from our group integrating MAS and OS-ssNMR techniques to give a comprehensive view of the biological processes occurring at cellular membranes. We focus on the main experiments for both techniques, with an emphasis on new implementation to increase both sensitivity and spectral resolution. We also describe how the structural constraints derived from both isotropic and anisotropic NMR parameters are integrated into dynamic structural modeling using replica-averaged orientational-restrained molecular dynamics simulations (RAOR-MD). We showcase small membrane proteins that are involved in Ca2+ transport and regulate cardiac and skeletal muscle contractility: phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa). We summarize our results for the structures of these polypeptides free and in complex with the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA, 110 kDa). Additionally, we illustrate the progress toward the determination of the structural topology of a six transmembrane protein associated with succinate and acetate transport (SatP, hexamer 120 kDa). From these examples, the integrated MAS and OS-ssNMR approach, in combination with modern computational methods, emerges as a way to overcome the challenges posed by studying large membrane protein systems.

Membrane Protein Structures in Lipid Bilayers; Small-Angle Neutron Scattering With Contrast-Matched Bicontinuous Cubic Phases.

This perspective describes advances in determining membrane protein structures in lipid bilayers using small-angle neutron scattering (SANS). Differentially labeled detergents with a homogeneous scattering length density facilitate contrast matching of detergent micelles; this has previously been used successfully to obtain the structures of membrane proteins. However, detergent micelles do not mimic the lipid bilayer environment of the cell membrane in vivo. Deuterated vesicles can be used to obtain the radius of gyration of membrane proteins, but protein-protein interference effects within the vesicles severely limits this method such that the protein structure cannot be modeled. We show herein that different membrane protein conformations can be distinguished within the lipid bilayer of the bicontinuous cubic phase using contrast-matching. Time-resolved studies performed using SANS illustrate the complex phase behavior in lyotropic liquid crystalline systems and emphasize the importance of this development. We believe that studying membrane protein structures and phase behavior in contrast-matched lipid bilayers will advance both biological and pharmaceutical applications of membrane-associated proteins, biosensors and food science.

Biophysical Basis of KDEL Receptor-Lipid Interactions in Secretory Signaling

KDEL receptor homologs 1-3 (KDELR1-3) are seven-pass membrane proteins involved in secretory homeostasis in Golgi and endoplasmic reticulum (ER). Binding of a semi-conserved Lys-Asp-Glu-Leu (“K-D-E-L”) chaperone sequence to KDELRs in the Golgi lumen initiates extensive membrane trafficking resulting in chaperone retrieval to ER and cargo release from Golgi. Aberrations in KDELR secretory signaling manifest in a wide range of diseases such as invasive cancers, dilated cardiomyopathy, neurodegeneration, and susceptibility to viral infections. At present, the role of anionic lipids in modulation of KDELR structure-function is not well understood. This is a major knowledge-gap as anionic lipids are crucial modulators of membrane dynamics, such as in the stabilization of integral membrane protein conformations and in recruitment of cytosolic signaling and trafficking proteins. In this investigation, we report interactions of KDELRs with a large panel of physiological anionic lipids that vary in headgroup composition and charge. Although KDELR homologs demonstrate differences in specificity for anionic lipids, KDELR1 and KDELR2, which share 94% sequence similarity, demonstrate broad interactions with anionic lipids enriched in the membrane cytosolic leaflet. KDELR3, on the other hand, demonstrates selectivity in interactions with anionic lipids. An in-silico Site-Identification by Ligand Competitive Saturation (SILCS) docking analysis of KDELR2 identifies multiple binding sites for anionic lipids. The residues in these sites are semi-conserved in KDELR homologs, thus providing residue-level insights into KDELR-lipid interactions. Molecular dynamics simulations demonstrate conformational modulation of KDELR2 by anionic lipids in a bilayer. The results generated in this investigation advance the biophysical and computational understanding of how membranes modulate KDELR conformations, thus controlling initiation of secretory trafficking.