CHARMM‐GUI Membrane Builder toward realistic biological membrane simulations

CHARMM‐GUI Membrane Builder, http://www.charmm‐gui.org/input/membrane , is a web‐based user interface designed to interactively build all‐atom protein/membrane or membrane‐only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance‐based algorithm for faster initial ion displacement, (5) CHARMM inputs for P₂₁ image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM‐GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. © 2014 Wiley Periodicals, Inc.     

A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non‐specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid‐binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent‐resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid‐II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non‐annular lipids based on their exchange rates in solution.     

Cofilin-Membrane Interactions: Electrostatic Effects in Phosphoinositide Lipid Binding

The actin cytoskeleton interacts with the cell membrane primarily through the indirect interactions of actin-binding proteins such as cofilin-1. The molecular mechanisms underlying the specific interactions of cofilin-1 with membrane lipids are still unclear. Here, we performed coarse-grain molecular dynamics simulations of cofilin-1 with complex lipid bilayers to analyze the specificity of protein-lipid interactions. We observed the maximal interactions with phosphoinositide (PIP) lipids, especially PIP₂ and PIP₃ lipids. A good match was observed between the residues predicted to interact and previous experimental studies. The clustering of PIP lipids around the membrane bound protein leads to an overall lipid demixing and gives rise to persistent membrane curvature. Further, through a series of control simulations, we observe that both electrostatics and geometry are critical for specificity of lipid binding. Our current study is a step towards understanding the physico-chemical basis of cofilin-PIP lipid interactions.     

Cytochrome‐P450‐Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide‐based lipid nanodiscs to “trap” the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein‐induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.     

Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions

The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene–maleic acid lipid particle (SMALP) technology can be coupled with hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics.     

Investigating the interactions of the first 17 amino acid residues of Huntingtin with lipid vesicles using mass spectrometry and molecular dynamics

The first 17 amino acid residues of Huntingtin protein (Nt17 of htt) are thought to play an important role in the protein's function; Nt17 is one of two membrane binding domains in htt. In this study the binding ability of Nt17 peptide with vesicles comprised of two subclasses of phospholipids is studied using electrospray ionization ‐ mass spectrometry (ESI‐MS) and molecular dynamics (MD) simulations. Overall, the peptide is shown to have a greater propensity to interact with vesicles of phosphatidylcholine (PC) rather than phosphatidylethanolamine (PE) lipids. Mass spectra show an increase in lipid‐bound peptide adducts where the ordering of the number of such specie is 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) > 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC) > 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3 phosphoethanolamine (POPE). MD simulations suggest that the compactness of the bilayer plays a role in governing peptide interactions. The peptide shows greater disruption of the DOPC bilayer order at the surface and interacts with the hydrophobic tails of lipid molecules via hydrophobic residues. Conversely, the POPE vesicle remains ordered and lipids display transient interactions with the peptide through the formation of hydrogen bonds with hydrophilic residues. The POPC system displays intermediate behavior with regard to the degree of peptide‐membrane interaction. Finally, the simulations suggest a helix stabilizing effect resulting from the interactions between hydrophobic residues and the lipid tails of the DOPC bilayer.     

Centerband‐only analysis of rotor‐unsynchronized spin echo for measurement of lipid 31P chemical shift anisotropy

Structural diversity and molecular flexibility of phospholipids are essential for biological membranes to play key roles in numerous cellular processes. Uncovering the behavior of individual lipids in membrane dynamics is crucial for understanding the molecular mechanisms underlying biological functions of cell membranes. In this paper, we introduce a simple method to investigate dynamics of lipid molecules in multi‐component systems by measuring the ³¹P chemical shift anisotropy (CSA) under magic angle spinning (MAS) conditions. For achieving both signal separation and CSA determination, we utilized a centerband‐only analysis of rotor‐unsynchronized spin echo (COARSE). This analysis is based on the curve fitting of periodic modulation of centerband intensity along the interpulse delay time in rotor‐unsynchronized spin‐echo experiments. The utility of COARSE was examined by using phospholipid vesicles, a three‐component lipid raft model system, and archaeal purple membranes. We found that the apparent advantages of this method are high resolution and high sensitivity given by the moderate MAS speed and the one‐dimensional acquisition with short spin‐echo delays. COARSE provides an alternative method for CSA measurement that is effective in the investigation of lipid polymorphologies. Copyright © 2015 John Wiley & Sons, Ltd.     

Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.     

Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high‐resolution shotgun lipidomics

The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high‐resolution shotgun lipidomics to characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high‐resolution Fourier transform mass spectrometry using an ion trap‐orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub‐ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we performed structural analysis using multistage activation on the ion trap‐orbitrap instrument as well as tandem mass analysis using a quadrupole time‐of‐flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described before. This uncharacterized lipid species features two head group structures composed of a trisaccharide residue carrying an uncommon sulfono group (?SO₃) and an inositol phosphate group. Both head groups are linked to a glycerol dialkyl glycerol tetraether core structure having isoprenoid chains with a total of 80 carbon atoms and 4 cyclopentane moieties. The shotgun lipidomics approach deployed here defines a novel workflow for exploratory lipid profiling of Archaea. Copyright © 2015 John Wiley & Sons, Ltd.     

Rhodopsin exhibits a preference for solvation by polyunsaturated docosohexaenoic acid

An all-atom molecular dynamics simulation of rhodopsin in a membrane environment has been carried out with lipid composition similar to that of the retinal membrane. The initial conformation of the protein was taken from the X-ray crystallographic structure (1F88), while those of the lipids came from a previous molecular dynamics simulation. During the course of the 12.5 ns simulation, the initially randomly placed lipids adopt an anisotropic solvation structure around the protein. The lipids, having one saturated stearic acid chain and one polyunsaturated docosohexaenoic acid chain with a zwitterionic phosphatidylcholine headgroup, arrange themselves to maximize contact between the polyunsaturated chain and the protein surface. This organization is driven by energetically favorable interactions between the transmembrance helices and the docosohexaenoyl chains that are largely of the van der Waals type. These observations are consistent with various experimental studies on rhodopsin and other G-protein coupled receptors and with the picture of extreme flexibility in polyunsaturated fatty acid chains that has arisen from recent NMR and computational work.     

G-Protein-Coupled Receptor–Membrane Interactions Depend on the Receptor Activation State

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of approximately one-third of currently marketed drugs. In particular, adenosine A₁ receptor (A₁AR) is an important therapeutic target for treating cardiac ischemia–reperfusion injuries, neuropathic pain, and renal diseases. As a prototypical GPCR, the A₁AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid–protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G-protein bound) A₁AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A₁AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A₁AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state. © 2019 Wiley Periodicals, Inc.     

Femtosecond Hydrogen Bond Dynamics of Bulk‐like and Bound Water at Positively and Negatively Charged Lipid Interfaces Revealed by 2D HD‐VSFG Spectroscopy

Interfacial water in the vicinity of lipids plays an important role in many biological processes, such as drug delivery, ion transportation, and lipid fusion. Hence, molecular‐level elucidation of the properties of water at lipid interfaces is of the utmost importance. We report the two‐dimensional heterodyne‐detected vibrational sum frequency generation (2D HD‐VSFG) study of the OH stretch of HOD at charged lipid interfaces, which shows that the hydrogen bond dynamics of interfacial water differ drastically, depending on the lipids. The data indicate that the spectral diffusion of the OH stretch at a positively charged lipid interface is dominated by the ultrafast (<～100 fs) component, followed by the minor sub‐picosecond slow dynamics, while the dynamics at a negatively charged lipid interface exhibit sub‐picosecond dynamics almost exclusively, implying that fast hydrogen bond fluctuation is prohibited. These results reveal that the ultrafast hydrogen bond dynamics at the positively charged lipid–water interface are attributable to the bulk‐like property of interfacial water, whereas the slow dynamics at the negatively charged lipid interface are due to bound water, which is hydrogen‐bonded to the hydrophilic head group.     

Lipid/protein interactions and the membrane/water interfacial region

Despite the importance of lipid/protein interactions in the folding, assembly, stability, and function of membrane proteins, information at an atomic level on how such proteins interact with the lipids that surround them remains sparse. The dynamics and flexible nature of the protein/bilayer interaction make it difficult to study, for example, by crystallographic means. However, based on recent progress in molecular simulations of membranes it is possible to address this problem computationally. This communication reports one of the first attempts to use multiple ns molecular simulations to establish a qualitative picture of the intermolecular interactions between the lipids of a bilayer and two topologically different membrane proteins for which a high resolution (2 A or better) X-ray structure is available.     

A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution.     

GridMAT‐MD: A grid‐based membrane analysis tool for use with molecular dynamics

GridMAT‐MD is a new program developed to aid in the analysis of lipid bilayers from molecular dynamics simulations. It reads a GROMACS coordinate file and generates two types of data: a two‐dimensional contour plot depicting membrane thickness, and a polygon‐based tessellation of the individual lipid headgroups. GridMAT‐MD can also account for proteins or small molecules within the headgroups of the lipids, closely approximating their occupied lateral area. The program requires no installation, is fast, and is freely available. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009     

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three‐dimensional structures. As lipids are dynamic by nature, their “structure” does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid‐raft‐related molecules, lipid–protein interactions, and membrane‐active natural products, we discuss current perspectives on membrane structural biology.     

Impact of Strong and Weak Lipid–Protein Interactions on the Structure of a Lipid Bilayer on a Gold Electrode Surface

A lipid bilayer deposited on an electrode surface can serve as a benchmark system to investigate lipid–protein interactions in the presence of physiological electric fields. Recoverin and myelin‐associated glycoprotein (MAG) are used to study the impact of strong and weak protein–lipid interactions on the structure of model lipid bilayers, respectively. The structural changes in lipid bilayers are followed using electrochemical polarization modulation infrared reflection–absorption spectroscopy (PM IRRAS). Recoverin contains a myristoyl group that anchors in the hydrophobic part of a cell membrane. Insertion of the protein into the 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphatidylcholine (DMPC)–cholesterol lipid bilayer leads to an increase in the capacitance of the lipid film adsorbed on a gold electrode surface. The stability and kinetics of the electric‐field‐driven adsorption–desorption process are not affected by the interaction with protein. Upon interaction with recoverin, the hydrophobic hydrocarbon chains become less ordered. The polar head groups are separated from each other, which allows for recoverin association in the membrane. MAG is known to interact with glycolipids present on the surface of a cell membrane. Upon probing the interaction of the DMPC–cholesterol–glycolipid bilayer with MAG a slight decrease in the capacity of the adsorbed lipid film is observed. The stability of the lipid bilayer increases towards negative potentials. At the molecular scale this interaction results in minor changes in the structure of the lipid bilayer. MAG causes small ordering in the hydrocarbon chains region and an increase in the hydration of the polar head groups. Combining an electrochemical approach with a structure‐sensitive technique, such as PM IRRAS, is a powerful tool to follow small but significant changes in the structure of a supramolecular assembly.     

Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

The Min proteins from E.coli position the bacterial cell‐division machinery through pole‐to‐pole oscillations. In vitro, Min protein self‐organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane‐enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell‐division protein FtsZ‐mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ‐mts structures on compartment size. Finally, co‐reconstitution of Min proteins and FtsZ‐mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self‐organization in confined volumes.     

Solid‐state NMR Study of the YadA Membrane‐Anchor Domain in the Bacterial Outer Membrane

MAS‐NMR was used to study the structure and dynamics at ambient temperatures of the membrane‐anchor domain of YadA (YadA‐M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA‐M protein in the E. coli lipid environment by using ¹³C‐¹³C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA‐M are unchanged relative to those in microcrystalline YadA‐M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition‐state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane‐embedded samples indicate greater flexibility of the ASSA region in the outer‐membrane preparation at physiological temperatures. This study will pave the way towards MAS‐NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.     

Lateral diffusion in lipid membranes through collective flows

There is no comprehensive model for the dynamics of cellular membranes. Even mechanisms of basic dynamic processes, such as lateral diffusion of lipids, are poorly understood. Our atomic-scale molecular dynamics simulations support a novel, concerted mechanism for lipid diffusion. We find that a lipid and its nearest neighbors move in unison, forming loosely defined clusters. What is more, the motions of lipids are correlated over tens of nanometers: the lateral displacements of lipids in a given monolayer produce striking two-dimensional flow patterns. These flow patterns should have wide implications, affecting, for example, the formation of membrane domains, protein functionality, and action of lipases and drugs on membranes.