Lipid-protein dynamics in biological membrane: A biophysical approach
At a glance:
ϖ The Plasma Membrane marks the boundary of a cell. Besides confining the cellular components, it is involved in numerous important cellular functions like signaling and vesicular transporting.
ϖ The lipids are building blocks of the PM who elicit their function by interacting with proteins. Lipids have a diverse structure providing a unique landscape for their protein counterpart to bind and perform their destined function.
ϖ These interactions are highly important to understand the complete picture of the processes. They are complex, tightly regulated and may involve divalent cations for effective interactions.
ϖ This review presents a promising approach to characterize these interactions with binding studies with Surface Plasmon Resonance (SPR) and Microscale Thermophoresis (MST), understand the lipid behavior at atomic level with Solid State Nuclear Magnetic Resonance (SSNMR) and eventually, predict the lipid-protein dynamics with Molecular Dynamics simulations.
ϖ These approaches can contribute immense knowledge towards lipid behavior by providing information about the lipid dynamics with structural evidence in NMR timescales. Since, we only had structural evidence of proteins these methods would result in a whole new field of lipid structural biology and complete the missing piece of the puzzle.
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Abstract
Characterization of membrane lipids binding to the proteins at the atomic level is still an untouched grey area in the field of structural biology. Over the years sophisticated techniques like X-ray Crystallography and Cryo-electron microscopy has provided immense structural information about the membrane proteins. A handful of these structures have revealed the proximity to their lipid partners in the membrane but a clear picture of how these lipid headgropus interact with their protein counterpart is still ambiguous. Since major biological processes like blood coagulation, apoptosis, cell signaling, intracellular trafficking and transporting channels are dependent on these intricate protein-membrane interactions and these proteins serve as drug targets, underscores the pressing needs of these studies. In this review, a brief outline of the experimental approaches taken to delineate membrane lipid-protein dynamics is presented. We propose a combination of biophysical, biochemical and computational methods that would help understand the dynamics of lipid behavior in biological membranes.
Introduction
The cellular plasma membrane (PM) is a bilayer structure. It comprises of neutral zwitterionic Phosphotidylcholine (PC) and Sphingomyelin (SM) on the outer leaflets and approximately 30% of total lipids are negatively charged lipids like Phosphotidylserine (PS), Phosphotidylethanolamine (PE) and Phosphotidylinositol (PI) mosly found on the inner leaflets 1. Localization of these lipids form microdomains in the PM which provides a suitable platform for the membrane proteins to execute functions like signaling, apoptosis, vesicular trafficking and blood coagulation.
The proteins interacting with the PM can be categorized as integral and peripheral membrane proteins. The integral membrane proteins are deeply embedded inside the hydrophobic core of the membrane whereas the peripheral membrane proteins are loosely attached to the membrane via electrostatic interactions. Predominantly the membrane lipid functions through the various interactions with the membrane proteins. These interactions could involve the acidic headgroup of the lipid interacting electrostatically with proteins involved in cell signaling cascade2 or sometimes via divalent cations like Calcium in case of the proteins involved in blood coagulation. Catering to wide array of biological functions the membrane proteins make up for one-third of the full proteome3 due to which 60% of the drug targets housing in the PM 4.
Lipids have complex structure. Due to polar head groups and hydrophobic backbone they are amphipathic in nature. This provides a polar environment on the outside and the hydrophobic environment on the inside of the PM. Dissecting further, lipids are made up of distinct chemical backbones made of glycerol in case of glycerophospholipids, isoprenes for sterols and sphingoid long chain bases for sphingolipids. The complexity is further increased by the saturation and unsaturation of the side chains of fatty acids attached to the backbone which determines the packing of the membrane. The lipids have charged and uncharged headgroups. Among which the anionic charged head groups generally serve as attractive interacting partners for the proteins to elicit their functions. To exemplify, during apoptosis the PS sequestered in the inner leaflet of the bilayer is flipped to the outer layer in the membrane which is then recognized by the Beta-2 glycoprotein 1 which helps the macrophages clear these cells 5. Proteins have adapted their structure to recognize these lipid moieties to elicit these functions.
This highly dynamic nature of the PM integrating varying protein structures and lipid molecules makes it challenging to study the lipid protein interactions at the atomic level. Additionally, the lipids lack long range order which makes it difficult to study with available diffraction methods. This review discusses the tools to solve the problem. We show how certain techniques at our disposal like binding studies using Surface Plasmon Resonance (SPR), Micro Scale Thermophoresis (MST) can help identify the lipid-protein partners suitable for Solid State NMR (SSNMR) studies to identify their interactions in NMR time scale and further predict their behavior with sophisticated MD simulations.
FUNCTIONAL LIPIDS AND THEIR INTERACTING PARTNERS
Different kinds of lipids contribute to the heterogeneity of the PM. However, among these some of them provide interactions for the proteins to elicit important biological role. These lipids can be called as functional lipids. For example, phosphotidylserine (PS) is a lipid with a negatively charged headgroup, is an important interacting partner of the C2 domain of Lactadherin. It binds stereospecifically to small amounts like 1% to 0.03% of lyso-PS in cells undergoing apoptosis6. With respect to functioning of Lactadherin PS would be characterized as a functional lipid.
The important aspect we aim to understand is the dynamic changes that occur during the interactions between the membrane protein and functional lipid over microseconds to seconds. Before moving forward with the appropriate solutions we first need to identify the different functional lipids anf their interacting partners.
There are two classes of interactions observed. Firstly, there are highly specific ones. These interactions are based on the stereospecificty of the lipid head groups. The pleckstrin homology domain of phospholipace C (a signaling protein in the MAPKinase pathway) binds very specifically to the phosphatidylinositol 4,5 bis phosphate (PtdIns(4,5)P2) 7. Secondly, there are non-specific binding which depends on the general physical properties like charge, amphiphilicity and curvature. The proteins which bind to membrane with lower specificity are the Gla domains which along with Calcium interacts with extracellular PS. There are annexin core domains which generally interacts with acidic phospholipids like PS and PE in presence of Calcium. In both the cases of Gla domains and the annexin core domains the intracellular levels of calcium play a crucial role wherein they interact via calcium bridging mechanism.(citation) With the help of transmission electron microscopy it was observed that α-Synuclein (α-Syn) is involved in membrane remodeling 8. This is a non-specific interaction which is unique in its function since the protein itself induces a structural change in the membrane.
ROLE OF CALCIUM IN SCLUPTURING THE PLASMA MEMBRANE
The cell strictly controls the levels of calcium asymmetrically. The reason being calcium is a secondary messenger sequestered in the sarcoplasmic reticulum. Upon receiving appropriate stimulus, the concentration of the calcium can increase from 0.01uM to 1uM in the cytoplasm whereas outside the cell it is maintained at a high concentration of 3mM9. The calcium is spatially separated from the anionic phospholipid PS. As mentioned before the PS is present in the inner leaflet of the membrane where calcium levels are generally low ~ 0.01uM. However, in case of the signaling pathway involved in lipid blood chemistry requires the proteins of the clotting cascade to interact with PS. These proteins have a patch of GLA residues which is known as gamma carboxyglutamate. The gamma carboxyglutamate is a post translationally modified form of the amino acid glutamate. The calcium forms a bridge between the carboxyl groups of the gla residues in the clotting proteins and the phosphate head groups of PS 10.Two long lived distinct population of PS head group was observed with two dimensional Solid State NMR spectroscopy (SSNMR) in presence of calcium. Possibly, this represents two different conformations of PS headgroups11 Further studies of PS headgroup conformation was observed in presence of Prothrombin as shown in (Figure 1). Unlike the previous study in this case a third population of PS conformation marked as PS3 between the alpha carbon and beta carbon was observed distinct from the other two PS1 and PS2 population12. Thus, we can observe that the calcium along with the protein is inducing a structural change on the membrane. MD simulation studies from unpublished data have shown that the calcium stays bound to the PS for upto 200 nanoseconds as compared to sodium which does not even last till 50 nanoseconds.
TECHNOLOGY TO STUDY BINDING OF MEMBRANE PROTEINS WITH LIPIDS
Surface Plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material stimulated by incident light. It is utilized for detecting molecular interactions. Binding of mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. This property is exploited in studying binding of proteins to lipids in real-time and can be used for both quantitative and qualitative measurements. It provides information on binding kinetics and affinity and can, in principle, be used for measuring dissociation constants (Kd) from sub nM to low mM. The equilibrium dissociation constant (Kd) can be derived from dissociation rate (‘off rate’, koff, kd) and association rate (‘on rate’, kon, ka) as shown in equation given below.
The Kd provides important information on the ongoing dynamic changes during the binding event. For studying the lipid-protein interaction, the lipids are immobilized on the gold plated sensor chip. The advantage of SPR is that it provides more flexibility with experimental conditions as compared to SSNMR. SPR requires label free protein and lipid samples, provides the option to work with various membrane compositions and allows studying both integral and peripheral membrane proteins. With SPR it is observed that the ζ subunit of T-cell receptor (TCR) generally unstructured in the cytoplasm gains defined structure upon contact with the anionic phospholipids. The study was carried out with different bilayer membrane compositions like 60:40 (POPC: POPG) and 70:30 (POPC: POPS) and even with cholesterol (DOPC/DOPS/cholesterol = 65:35:3) 13.
Microscale thermoporesis (MST) is a technology for the interaction analysis of biomolecules. It is based on thermopheresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell or conformation. The technique is highly sensitive to virtually any change in molecular properties, allowing for a precise quantification of molecular events independent of the size or nature of the investigated specimen. Notably, MST is a label free binding study, which detects changes in the hydration shell of the lipid and the protein and allows studying these interactions in a buffer of our choice. The sample is subjected to a temperature gradient with help of infra-red light resulting in the movement of molecules known as thermophoresis. The method is robust, requires sample in the nanomolar scale, is easy to optimize and is label free. This technology is complementary to surface-based sensors like SPR.
MEMBRANE MODELS
Liposomes and Nanodiscs are widely used membrane models for lipid protein interactions. The liposomes can be made up from small (0.025um) to large (2.5um) vesicles and provides us with large opportunity for choosing the binding partners 14. Nanodiscs (ND) are lipid bilayers of 8-16nm diameter and the size is determined by the membrane scaffold proteins and the stoichiometry of the lipids 15. These (ND) are self-assembly systems and provide us with a larger surface area for protein binding studies. With both these systems we can use varying compositions of phospholipids to simulate an in vivo niche. These membrane models allow us to mimic the membrane curvature by introducing PE in the system. This is important for certain proteins like VPS20 which senses the membrane curvature for its activity 16
IDENTIFYING THE LIPID HEAD GROUP FUNCTION
The lipid headgroups are made of functional groups like ammonium, hydroxides and phosphates. The functional groups might contribute to the lipid-protein interaction. However, these interactions have not been characterized yet. A novel approach to study these interactions would be to synthesize analogs for the lipid headgroups. These analogs have the same functional groups but different conformation (Figure 2). Binding studies with analogs can shed light on the importance of these functional groups that might be involved in these interactions.
PREDICTING THE LIPID BEHAVIOUR WITH MD SIMULATIONS
MD simulation is a great tool to resolve the structural and mechanistic basis of lipid-protein interaction and have been widely used for this purpose. But the complex lipid backbone structure causes their slow lateral diffusion across the membrane increasing the time scale to study the entire system. Previously popular coarse-grained lipid models17 are static in nature and does not offer a mobile membrane environment which is important in understanding the lipid-protein interaction as these lipids diffuse through membrane over time. The ligand binding induces structural change on the lipid head-group which is difficult to observe with the coarse grained model. To overcome these problems, a Highly Mobile Membrane Mimetic (HMMM) has been devised.
HMMM replaces the long hydrophobic chains of the lipids with organic solvent (Figure 3) which increase the lateral diffusion by 1-2 orders range of magnitude 18. As a result, the time-scale to simulate the whole system reduces and as compared to conventional simulating models allowing higher sampling number. The HMMM has shown to be effective in studying both the peripheral membrane proteins like the coagulation proteins19, cytochrome P45020, hemoglobin N21, and the integral membrane proteins like synaptogamin 22
Conclusion
Integration of the binding studies with solid state NMR and MD simulation is what we need to answer the important question of how lipids interact with proteins. The real-time analysis of binding event between the protein and lipid can help us to narrow down very specific protein-lipid partners which can be studied with SSNMR. The aim is to find a stable lipid-protein complex, their lipid head groups and specific amino acid groups respectively, involved in these interactions. SPR and MST are two techniques which can help in identifying an appropriate lipid-protein complex which can be further resolved with SSNMR at the atomic level. Combining the spectral data from SSNMR with MD simulations in HMMM will give a clear picture of these interactions and predict their behavior in longer time scale which is not feasible with any biophysical technique. Combination of these techniques will potentiate the field of lipid structural biology and open avenues for exploring membrane protein interaction in numerous biological processes.