Nanodiscs: A Controlled Bilayer Surface for the Study of Membrane Proteins

Abstract

The study of membrane proteins and receptors presents many challenges to researchers wishing to perform biophysical measurements to determine the structure, function, and mechanism of action of such components. In most cases, to be fully functional, proteins and receptors require the presence of a native phospholipid bilayer. In addition, many complex multiprotein assemblies involved in cellular communication require an integral membrane protein as well as a membrane surface for assembly and information transfer to soluble partners in a signaling cascade. Incorporation of membrane proteins into Nanodiscs renders the target soluble and provides a native bilayer environment with precisely controlled composition of lipids, cholesterol, and other components. Likewise, Nanodiscs provide a surface of defined area useful in revealing lipid specificity and affinities for the assembly of signaling complexes. In this review, we highlight several biophysical techniques made possible through the use of Nanodiscs.

INTRODUCTION TO NANODISCS

Multiple cellular processes make use of biological membranes for function. Membrane proteins constitute a large fraction of all proteins and can adopt a variety of topologies with respect to the phospholipid bilayer. Many systems contain integral membrane proteins, wherein much of the structure is contained within the bilayer, with the polypeptide often making multiple passes. Examples include the seven-transmembrane (7-TM) G protein–coupled receptors, multisubunit transporters, and ion channels as well as complex machines such as the photosynthetic reaction center, cytochrome oxidase, and drug efflux pumps. Other membrane proteins may have a larger part of their structure exposed to the aqueous environment with a portion of the polypeptide chain embedded in the membrane. Examples include cytochrome P450 and its electron transfer partners, flavoprotein reductase, and cytochrome b₅. Yet another class of proteins may make use of simple organic or polypeptide tethers to aid membrane association, often also relying on electrostatic or specific ionic interactions between phospholipid head groups and the protein. In many cases, phospholipids contribute critical recognition components and/or active site determinants that are important for structural integrity and full functionality of the membrane protein.

In general, all three classes of membrane proteins have presented challenges when studied using standard biophysical and biochemical techniques for investigating the structure and function of soluble proteins. Removing the membrane protein from its phospholipid environment often results in aggregation and inactivation. Over the years, many solutions, including incorporation of target proteins into liposomes and solubilization with detergents, amphipols, or SMALPs (styrene–maleic acid lipid particles), have been utilized (39). A recent approach enjoying wide popularity is self-assembly of membrane protein or complex into Nanodiscs, an optimized form of discoidal lipoprotein particle.

Nanodiscs are discoidal lipid bilayers that can be engineered to have varying diameters (∼6–16 nm), which are stabilized and made soluble in aqueous solutions via two encircling amphipathic helical protein belts termed membrane scaffold proteins (MSPs) (10, 36, 55). Nanodiscs resemble the transient form of high-density lipoprotein particles investigated in detail by the atherosclerosis community for several decades (20, 97). The Nanodisc system has been extensively reviewed, including in an exhaustively referenced article published in Chemical Reviews in 2017 (40). The desire to optimize the size and homogeneity of Nanodiscs led to genetic engineering of Apo-AI (apolipoprotein A-I), the main protein component of high-density lipoprotein, and yielded several variants of defined size containing a variety of tags (40, 95). Nanodisc size is determined by the length of the MSP belt and the number of lipids needed to fill the encircling rings. The MSPs most commonly used worldwide include MSP1D1, which generates Nanodiscs ∼9.7 nm in diameter, and MSP1D1E3, in which three additional 22-mer helices are inserted to generate discs ∼12 nm in diameter (36). A shortened MSP, recently pioneered by Wagner and colleagues (55), is made by deleting one 22-mer helix and generates smaller discs that have been useful for solution nuclear magnetic resonance (NMR) studies.

Nanodisc technology is now extensively utilized. Indeed, a search of the literature using the keywords “Nanodiscs–membrane protein” returns more than 800 papers from laboratories throughout the world. Such wide use has been facilitated by the ready availability of genes expressing numerous MSPs from the depository AddGene^® (more than 1,400 samples shipped) as well as purified MSPs from commercial chemical vendors (e.g., Sigma-Aldrich).

NANODISC ASSEMBLY AND STRUCTURE

The term Nanodisc refers herein to a true phospholipid bilayer with an encircling amphipathic helical protein, the MSP. The length of the MSP provides a metric for the formation of particles with defined diameter, as opposed to bicelles, which can form particles of varying size dependent on the ration of short- and long-chain lipids. With a single lipid component, the bilayer exhibits a classic phase transition of the core, although the small number of lipids present broaden the transition from that of an infinite planar membrane (37).

Detergent Removal Initiates Self-Assembly

Nanodiscs are formed by self-assembly from detergent-solubilized components in a process similar to the formation of reconstituted high-density lipoprotein particles described by Jonas and others (14, 36, 63, 95). The chosen MSP together with the desired lipid, or synthetic lipid mixture, is solubilized in detergent. Removal of the detergent, either by extensive dialysis or by adsorption onto hydrophobic beads, initiates self-assembly and the formation of discoidal bilayers of uniform size and dispersivity. If the stoichiometry of MSP:lipid is exactly correct, only discs of a single size are formed. If the ratio is off, aggregates of excess lipids and/or MSPs may form along with the Nanodiscs. Underlipidation generates less stable ellipsoidal bilayers (104). This process of Nanodisc formation, as well as its detailed protocols and documentation of the correct stoichiometry for various synthetic lipids and mixtures, has been extensively reviewed in recent literature (39, 40, 54, 77, 95, 99).

Structural Characterization of Nanodiscs

Nanodiscs formed from the self-assembly process can be characterized by a wide variety of biophysical methods. The fidelity of the initial self-assembly reaction is easily verified by size-exclusion chromatography, which displays a single symmetric elution peak under the correct MSP:lipid stoichiometry (40). Solution X-ray scattering of Nanodiscs is an important tool used to define the size and shape of the lipoprotein particle (36, 104). Early work used atomic force microscopy to image these discoidal lipoprotein particles on atomically flat mica (13) and revealed heterogeneity in size when full-length Apo-AI was used as a scaffold. This also led to the realization that precise control of the tip feedback loop used in atomic force microscopy could also fuse and pattern bilayers (25). Precise height measurements of individual Nanodiscs as well as the dimensions of incorporated membrane proteins were obtained by constructing calibrated force-distance cantilevers (8, 9, 13). Evolution of corresponding instrumentation, including fine-tip dimensions and automatic force feedback, now allow routine high-resolution imaging of Nanodiscs on flat surfaces.

Nanodiscs can be directly visualized by electron microscopy (EM), although negative staining demands careful attention to artifacts induced by the stain and to possible preferences for orientation on the grid depending on the materials used. Another concern is the potential interaction of the cationic stain that can bridge multiple Nanodiscs, giving rise to rouleaux or stacking of the bilayers like a sleeve of quarters (45). Recent high-resolution imaging of integral membrane proteins in Nanodiscs using cryo-EM points to increased use of this method to investigate the structure of membrane proteins and complex assemblies in a bilayer environment (1, 43, 46).

Nanodiscs are widely used to investigate membrane proteins by magnetic resonance spectroscopy. Initial successes utilized solid-state NMR (64, 65, 73, 88). The use of Nanodiscs in solution NMR has exploded in recent years (see 40). Noteworthy is the complete structure determination of the integral membrane protein OmpX by Hagn & Wagner (56) using engineered smaller scaffold proteins (55). Other resonance methodologies such as EPR, DEER, and ENDOR have also revealed insights into membrane proteins when the target is assembled into Nanodiscs (111). Mass spectrometry efforts by Gross, Robinson, Marty, and colleagues (40, 60, 80, 84) have shown how to precisely characterize lipid composition and to identify incorporated membrane proteins of Nanodisc assemblies. Recent success in forming soluble membrane protein libraries (83) by moving a tissue membrane proteome into individual Nanodiscs promises to have a major impact in high-throughput screening of membrane protein targets in pharmaceutical discovery (114).

Physical Properties of Nanodiscs

Characterizations of Nanodiscs utilize a wide variety of biophysical and structural techniques, which have been applied to incorporated membrane protein targets as well as peripheral membrane proteins interacting with the bilayer surface. Many of these techniques have been recently reviewed and are not further described in detail in this section. These include pulsed magnetic methods such as double electron–electron resonance, cryo-EM, NMR, spectroscopic tools such as circular dichroism and optical waveguides, and a variety of scattering methods using X-rays, gamma rays, and neutrons.

Lateral size and bilayer thickness.

The overall size of the Nanodisc bilayer is determined by the length of the encircling MSP belt. As described by Denisov et al. (36), carefully measuring the number of lipids verses length of the amphipathic helical protein at various lipid stoichiometries allows for documentation of regions involved in determining overall diameter. Nanodisc height, ∼5.5 nm, is precisely quantitated by solution X-ray scattering, atomic force microscopy, and cryo-EM. Small-angle X-ray scattering also documents the mean area per lipid head group and its variation with temperature (37). The phase transition behavior of synthetic lipid bilayer Nanodiscs has also been measured by fluorescence and differential scanning calorimetry (102). The physical parameters of various Nanodisc preparations needed for subsequent discussion are reviewed in our recent Chemical Reviews article and elsewhere (40, 62).

Electrostatic charge of nanodiscs.

Nanodiscs can be self-assembled with a plethora of lipid types and synthetic lipid mixtures as well as a composition that mimics in vivo tissues. This opens the door to investigating the formation of protein-membrane binding events and the formation of larger multiprotein signaling complexes on the bilayer surface. Important in such studies is the ability to form Nanodiscs with varying lipid compositions, particularly with a defined number of charges. This allows understanding of not only the electrostatic component for the recruitment and overall affinity, but also any specificity due to head group structure.

Using small-angle X-ray scattering, Shaw (101) generated and characterized the increasing numbers of anionic and cationic lipids of Nanodiscs. Although the ∼10-nm-diameter disc could be formed with >90% anionic lipid POPS, self-assembly of Nanodiscs failed with phospholipids containing more than 15% tertiary amine head groups. Placing 50–100 charges within a 10-nm circle creates significant electrostatic interactions. With a titratable side chain, solvent protons and cations neutralize the overall charge. In addition, the high charge density recruits a corresponding ion cloud modifying the total electrostatic picture. Nanodiscs are thus polyelectrolytes, and a precise measurement of the Debye-Huckle-Henry charge is needed. Membrane-confined electrophoresis, pioneered by Laue et al. (69), together with analytical ultracentrifugation has been used to document the actual charge on Nanodiscs of various sizes and lipid compositions (59).

Self-Assembling Membrane Proteins into Nanodisc Bilayers

Incorporation of membrane proteins into Nanodiscs has become a standard method for revealing the structure and documenting the function of membrane protein targets. Including a membrane protein target into the detergent-solubilized MSP and lipid and then removing the detergent results in self-assembly of the target into the bilayer in a naturalistic presentation that preserves activity and structure. Detailed procedures have been extensively described in recent reviews and methods chapters (14, 36, 95, 99) and are not discussed here to any great extent. The Nanodisc system has been very valuable in investigating 7-TM signaling molecules such as the G protein–coupled receptors (11, 12, 15, 72); complex chemotactic receptors such as the trimer of dimers (18); the intact secretory translocase complex (2, 30, 31); numerous cytochrome P450s (38); many protein electron transfer complexes (5); and complex machines such as the photosynthetic reaction center (57), cytochrome oxidase (91), and numerous transporters and channels (35, 40, 44, 79, 100).

Activity and Stability of Membrane Proteins in Nanodiscs

From the perspective of the membrane protein target, the true bilayer of the Nanodisc feels much like a native environment, and full activity and functionality of the target are realized. Such is not always the case for other entities, such as SMALPs (70) and amphipols (92), that act more like detergents in solubilizing the membrane protein. In many cases, the target is stabilized against thermal, pressure, or cosolvent perturbations. Being in the correct configuration with respect to the bilayer leads to an epitaxial presentation of a potential antigenic surface that is finding important applications in the rapid production of vaccines (17) and monoclonal antibodies (42).

Providing further stability, the Nanodisc can also be viewed as a cassette that allows surface-based, solution, and gas-phase analytical assay methods to be readily used without fear of target denaturation or poisoning of the sensor surface. Thus, planar (88), localized surface plasmon resonance (SPR) (33, 121, 122), resonance Raman spectroscopy (78), linear optical waveguide (16), silicon ring resonator (105), and electrochemistry (32) can be utilized. Given the stability of incorporated proteins in Nanodiscs, mass spectrometry is also used to characterize the target (80, 84). Mass spectrometry of the bound lipids provides tools for membrane proteomics of soluble membrane protein libraries made directly from tissue (60, 81, 83, 114).

NANODISCS AS CONTROLLED SURFACES FOR THE RECRUITMENT AND FORMATION OF SIGNALING COMPLEXES

The high degree of homogeneity of correctly assembled Nanodiscs means that a membrane surface of defined composition and area can be created. Because the Nanodisc enables use of multiple surface- and solution-based assays without inactivation of the membrane protein target, protein-protein and protein-lipid interactions may be studied directly. Indeed, the membrane surface is the cellular site for the recruitment of many signaling systems: We now realize the membrane plays a key role in determining the active structure of individual signaling molecules as well as correctly forming specific multiprotein complexes required for the control of complex pathways.

For the remainder of this review, we focus on recently acquired data on three very different signaling systems where the interaction of proteins with a Nanodisc of defined size and lipid composition can be precisely quantitated. First, we discuss protein-protein and protein-lipid interactions that modulate human blood coagulation events. Second, we review efforts to understand the membrane interactions involved in integrin activation. Third, we explore the recruitment of a small GTPase to the membrane surface and the formation of multicomponent complexes involved in cancer signaling.

Analytical Methods for Measuring Protein Binding to Nanodiscs

All the three systems discussed prefer measurement of the binding of a protein or protein complex to the membrane surface of a Nanodisc. Over the past decade, numerous methods have been used to monitor such association events. In particular, SPR has long been used to monitor macromolecular associations. Because SPR is a surface phenomenon, Nanodiscs need to be affixed or tethered at or near the surface. Numerous systems use readily available biotinylated, His-tagged, FLAG-tagged, nucleic acid–labeled, or covalently linked MSPs (52). Boundary diffusion issues complicate analysis and introduce a challenge to using SPR, although analyte gradient tricks partially alleviate the problem (82). We describe this assay method in determining how blood coagulation proteins associate with Nanodiscs.

Gold or silver nanoparticles generate a localized SPR. Where a chromophore on the target or Nanodisc has an absorption band near the localized SPR, a dramatic enhancement of the signal occurs. For instance, highly accurate monitoring of small-molecule binding to cytochrome P450 in Nanodiscs using this approach was 1,000-fold more sensitive than normal SPR (121, 122). Changes in the solution dielectric constant around a silicon/silicon oxide ring resonator have provided parallel detection of several analytes binding to Nanodisc bilayers and incorporated membrane proteins (105). Mass detection via resonant cantilevers has been used with affixed ganglioside Nanodiscs to monitor cholera toxin binding (108). Several investigators have also successfully monitored small-molecule and macromolecule association using isothermal titration calorimetry (86).

Fluorescence resonance energy transfer (FRET) is a widely used technique to monitor binding events. The Nanodisc system is an ideal platform for FRET measurements because the encircling MSP hosts a unique labeling site for attachment of donor/acceptor molecules. We describe this approach in the following discussion of talin binding (see the section titled Integrin signaling).

A downside of FRET, however, is the requirement that the associating protein be labeled; a label-free solution-based assay would be ideal. Here, the Nanodisc system also offers a unique opportunity. The discoidal bilayer of ∼5.5-nm height and ∼10-nm diameter has two principal axes of rotation (see Figure 1). Rotational correlation times (τ _R) can be measured by fluorescence anisotropy decay in the time domain or by multifrequency phase fluorimetry (67) via an attached fluorophore. For highest sensitivity, the label should have a fluorescent lifetime near the rotational time. The average τ _R of 10-nm Nanodiscs as measured by NMR is roughly 70 ns (55, 93). One solution is to use transition metal chelates such as ruthenium. The tris(bipyridine) (RuBiPy) nucleus offers lifetimes on the order of 550 ns, high quantum yield, and inert chemical reactivity. A single amine reactive group on one of the bipyridal groups is commercially available, and several reports of cysteine-specific labels are available (25a, 68, 100, 109a). For example, a RuBiPy label has been attached to a genetically engineered cysteine residue, or an amine, on the MSP. Rotational correlation times were measured by fitting the frequency-dependent phase shift between vertical and horizontally polarized emission (67) and yielded ∼20 ns and ∼100 ns corresponding to the two principal axes shown in Figure 1. The challenge here is detecting the binding of a small protein, such as the oncogene KRas4b (∼21 kD), to the Nanodisc. Monitoring the phase shift at a single frequency, which corresponds to the maximum change on protein binding, gives highly accurate and reproducible results (53). This approach has been used successfully in measurements of KRas4b binding to Nanodiscs of varying anionic lipid compositions.

Figure 1.

Schematic illustration of Nanodiscs showing the encircling membrane scaffold proteins (blue) and phospholipids with head groups (red ). Dimensions shown correspond to MSP1D1 with a bilayer thickness of ∼5.5 nm and diameter of ∼10 nm. Rotational motion of the Nanodisc in solution along its principal axes is indicated.

Three Examples of Multiprotein Associations on Nanodisc Surfaces

The use of Nanodiscs as a membrane surface of precisely controlled size and lipid composition has become increasingly popular. Intra- and intercellular communication involves membrane proteins, and the complex signaling cascade often involves formation of multiprotein complexes on the membrane surface. Likewise, many enzymatic cascades involve a membrane protein that provides affinity and selectivity for circulating proteins engaged in amplification in response to injury or other signaling processes. Indeed, in many cases, anionic lipids play a special role in the recruitment of signaling proteins to the membrane surface. Understanding the detailed mechanisms of this interaction requires a system wherein the composition and concentration of the membrane can be controlled. The Nanodisc system has proved ideal for these investigations. We examine three separate systems where different analytical methods have been used to quantitate such binding interactions.

Blood coagulation cascade.

Controlling bleeding is vital to the health of an organism. Complex pathways exert control over the formation of a blood clot. The clotting cascade requires assembly of protease-cofactor complexes on membranes with exposed anionic phospholipids. A suitable surface typically contains a mixture of neutral and anionic phospholipids, such as may be encountered following platelet activation or cell/vascular damage. This process has been investigated for many years, with the understanding that many anionic lipids can stimulate clotting reactions among which phosphatidylserine (PS) head groups are the most active (89, 109, 124).

The details of how anionic membrane surfaces accelerate clotting reactions are not known. However, multiple explanations have been offered. Among these is an increase in the local concentration of reactants that induces specific conformational changes of the protein cofactors and restricts movement of proteins to two dimensions. The Nanodisc system, together with measurements using SPR, has provided an excellent example of how such molecular information is obtained. The binding of factor X and factor IV to Nanodiscs of defined anionic lipid composition were monitored by SPR, which separated head-group specificity from overall electrostatic interactions (89, 109, 124). In this case, the interaction with negatively charged γ-carboxyglutamate residues (Gla domains) and anionic PS head groups is made by bridging divalent cations. Calcium ions induce anionic phospholipids to cluster, with the clotting proteins assembling preferentially on anionic lipid-rich microdomains that are found, for example, on the exterior of the vasculature. A challenge has been to develop a means of controlling the partitioning of clotting proteins with membrane microdomains. Vesicles did not work because the lipids are mobile and can “cap,” thereby changing the local environment at the protein-membrane interface. Nanodiscs have allowed us to probe with nanometer resolution how local variations in phospholipid composition regulate the activity of these key protease-cofactor complexes. Furthermore, structural information can be gleaned using new developments in solid-state NMR measurements (64).

Integrin signaling.

Integrins play a central role in the dynamics of cell adhesion and cell-cell interactions. They are involved in a wide range of biological processes, including embryonic development, hemostasis, cell migration, wound healing, and the immune response. Their impaired function has been linked to key human diseases such as arthritis, heart attack, stroke, and cancer (61). Integrins are membrane-bound, heterodimeric adhesion receptors consisting of one α and one β subunit, each possessing a large extracellular domain, a TM domain, and a small cytoplasmic domain. Their function is complex and involves interactions with many proteins, resulting in a mechanical link of the extracellular matrix to the cytoskeleton (120, 123). The inactive state of integrin has a compact or bent extracellular domain with the TM helices of the α and β subunits tightly associated (75). The active state may have an extended extracellular domain with the TM helices separated and no longer in contact with each other (76). One mechanism of activation, known as inside-out signaling, involves the interaction of adapter proteins with the small cytoplasmic domain of the β integrin resulting in conformational changes in the extracellular domain and an increased affinity for ligands. Talin, a large 270-kDa actin-binding protein, is a key player in inside-out signaling (22, 41, 117).

Talin is the key activator of integrins. It is also one of several adapter proteins involved in linking integrins to the actin cytoskeleton (3, 21, 28). Talin consists of a head domain (THD) [400 amino acids (aa)], a linker region (80 aa), and a rod domain (2,060 aa) (90, 94). The THD contains a FERM (band 4.1; Ezrin, Radixin, Moesin homology) domain consisting of F0, F1, F2, and F3 subdomains. Activation of integrin by talin is primarily mediated by the phosphotyrosine binding (PTB)-like domain (F3) (4, 23, 24, 47). The THD interacts with the cytoplasmic tail of β integrin in two steps: First, a membrane distal sequence is bound; second, the membrane proximal helix of β integrin interacts (110, 112, 113) with the negatively charged surface of the membrane. A basic patch located on the F2 domain, known as the membrane orientation patch, may mediate binding to anionic regions of the membrane and orient the head domain on the bilayer for efficient interaction with the integrin cytoplasmic domain (4, 96).

Talin may exist in multiple conformations within the cell—for example, a monomer-dimer equilibrium form manifested by the interaction of a short C-terminal dimerization domain and an autoinhibited form (49, 51, 87). In the inhibited form, a segment of the talin rod domain (residues 1,654–2,344) binds to the PTB site of the F3 subdomain, preventing interaction of the THD with integrin, the phospholipid membrane, or both (50). A key step in talin activation is relieving the interactions stabilizing the autoinhibited state, thus exposing PTB on the THD. Several factors, including the binding of adapter proteins and interaction with PIP2 (phosphatidylinositol 4,5-biphosphate) bilayers, activate talin (24, 34, 41, 47, 71, 96, 116). Of particular interest is talin interaction with PIP2. PIPKIγ (phosphatidylinositol 4-phosphate 5-kinase type 1 gamma) is responsible for the generation of PIP2 from phosphatidylinositol 4-phosphate and may be targeted to the membrane surface and localized to focal adhesions through interaction with talin (7, 34, 41, 74). In fact, talin binding increases the kinase activity of PIPKIγ severalfold (41). Importantly, these signaling interactions occur on a membrane surface; included among these are interactions with integrin, as an integral membrane protein, as well as the recruitment of activating molecules such as talin. The membrane-talin interaction may involve ionic bridges from basic residues on talin with anionic lipids. Understanding the molecular details of these specific salt-bridge interactions is critical in describing the mechanisms of inside-out signaling. Here, Nanodiscs provide the perfect platform, yielding a membrane surface of defined area and composition where a plethora of analytical tools can be used to quantitate the thermodynamics and kinetics of the interaction to reveal any head-group specificity critical for function.

Ye and coworkers (116) first reported using Nanodiscs to study integrin activation. Integrin α_IIbβ₃ was incorporated into Nanodiscs containing a 1:1 ratio of DMPC (dimyristoyl phosphatidylcholine):DMPG (dimyristoyl phosphatidylglycerol) lipids. This membrane mimetic successfully recreated the final step in integrin activation. Binding of the antibody PAC I, which is specific for active integrins, showed that the TDH is sufficient for activation of integrin embedded in phospholipid bilayers. EM images of integrin Nanodiscs in the presence of talin showed a marked change in the “outside” integrin topology, indicating an extended extracellular domain that could interact favorably with the extracellular matrix. Activity was further confirmed in EM images of Nanodiscs showing a significant increase of fibrin-associated Nanodiscs in the presence of talin (116).

Since the first publication of integrin-embedded Nanodiscs, the structure of membrane-associated integrin has been further refined using EM. Choi and coworkers (26) used EM images of Nanodiscs harboring integrin α_IIbβ₃ to show that the membrane-embedded structure differs significantly from the solution structure of the extracellular domain, primarily in the topology of the TM helices and the orientation of the ligand binding domains. In another study, negative-stained images demonstrated that ligand-free Mn²⁺-activated integrin exists in a compact form. However, in the presence of a physiological ligand, integrin extends to 18–20 nm, suggesting that ligand binding also plays a critical role in the conformational equilibrium (29). Cryo-EM has been used to examine an integrin bound to Nanodiscs and revealed four distinct conformational states (115): a compact or bent inactive state, an extended active state, and two intermediate states. Addition of talin or ligand strongly favors the active extended conformation (115).

In addition to the groundbreaking structural studies of membrane-embedded integrin, Nanodiscs have been used to develop sophisticated fluorescence-based techniques that probe both binding and structural properties of integrin signaling complexes. A critical step in integrin activation is disruption of the interaction between the TM helices of the α and β subunits, leading to extension of the extracellular domains. The Ginsberg lab has developed a novel assay to assess the tilt angle of the β TM helix by labeling the helix at the inner or outer bilayer interface with environmentally sensitive dyes (66). When the labeled β TM domains are incorporated into Nanodiscs, talin binding to the cytoplasmic domain promotes the burying of both the inner and outer fluorophores attached to the TM helix. This can happen only if the tilt angle of the TM domain with respect to the bilayer is altered. Mutations that introduce flexibility within the TM helix abolish helix tilting. The change in TM helix topology likely favors disruption of the interaction between the α and β subunits of the TM helix, thus resulting in activation.

FRET has also been developed to measure the interaction of talin with bilayer surfaces. A cysteine mutation in the MSP1D1 belt at position 73 (D73C) allows for site-specific labeling of the Nanodiscs with a sulfhydryl-reactive fluorophore. Nanodiscs prepared with a fluorescently tagged MSP can be used as a substrate for measuring the binding of peripheral membrane proteins that have been labeled with a quencher or a FRET acceptor (Figure 2a). Ye and coworkers (118) used a FRET-based assay to investigate the lipid specificity of talin for bilayers containing DMPC, DMPS (dimyristoyl phosphatidylserine), DMPG, DMPA (dimyristoyl phosphatidic acid), and PIP2. They showed that factors beyond total charge give rise to a specificity of talin for PIP2-containing membranes. This is most easily seen by using a FRET-based fluorescence quenching assay to monitor binding free energy. The ability to determine the precise charge on a Nanodisc via membrane-confined electrophoresis (see the previous section for a discussion) enables the dependence of the binding free energy on the actual Nanodisc charge (Figure 2b), determined at various percentages of anionic lipid in a DMPC background. If affinities were based solely on bilayer charge, the free energies should have similar charge dependencies, which is clearly not the case.

Figure 2.

(a) Fluorescence resonance energy transfer between MSP tagged with tetramethylrhodamine and THD labeled with Uniblue A as the soluble protein binds charged lipids in the Nanodisc. (b) Binding free energy as talin binds to Nanodiscs prepared with varying DMPS or PIP2 content. Total charge on the Nanodisc is plotted on the abscissa. Abbreviations: DMPS, dimyristoyl phosphatidylserine; MSP, membrane scaffold protein; PIP2, phosphatidylinositol 4,5-biphosphate; THD, talin head domain; Z_DHH, Debye-Huckle-Henry charge.

In addition, calculated Förster distances show that PIP2 promotes a conformation of the THD that places the integrin binding domain (F3) 10 Ǻ closer to the bilayer surface compared with binding PS-containing bilayers, thus implicating PIP2 as a regulator in integrin activation. In a subsequent study, Nanodiscs were used to investigate the ability of PIP2 to activate the autoinhibited form of talin (119). The results show that binding of talin to PS-containing membranes, when performed in the presence of the purified rod domain subunits, is drastically inhibited, and this inhibition follows a classical competitive inhibition model. In contrast, the binding of talin to PIP2 membranes is relatively unaffected by the presence of the inhibitory domain, thereby implicating PIP2 as a key player in talin activation. Thus, measurements of talin membrane interactions on a controlled bilayer surface have given rise to new insights into talin-mediated integrin activation.

Nanodiscs and the oncogene signaling cascade.

Signaling pathways involved in the control of cell growth and proliferation initiate at a membrane surface. Ligand binding through specific receptors, such as the receptor tyrosine kinases, transmits information across the membrane, where a group of signaling proteins interacts to communicate with various effector molecules.

Ras proteins, so-called owing to the discovery in the 1960s of their involvement in rat sarcomas, are small GTPases essential to multiple signaling pathways responsible for cell growth, proliferation, and survival in many organisms (6). These proteins bind guanine nucleotide and are “off “ when GDP is bound and “on” when GTP is at the active site. Signal transduction through these ∼21-kDa proteins is activated by guanosine exchange factors, which assist with GTP replacement of GDP (19). Nucleotide binding induces a conformational change in the switch I and switch II regions of Ras that permits the recruitment, association, and activation of downstream kinases that control cellular function. Transition from the GTP-bound “on” state to the GDP-bound “off “ state occurs when Ras interacts with GTPase-activating proteins, which are thought to act by stabilizing the GTP to GDP transition state (98). In a healthy cell, Ras activity is tightly regulated by the actions of its upstream effector proteins. The “on” state drives the signaling pathways that induce cell migration and growth. Mutants that favor the “on” conformation are commonly found in human cancers. Indeed, roughly 20% of all tumors have activating Ras mutations that effectively decouple the output of this protein from upstream signaling events. Point mutations that generate constitutively active Ras are especially common in several forms of cancer, including pancreatic cancer, in which 90% of KRas isoforms have activating mutations (103). Despite decades of research, pharmaceutical intervention in the Ras pathway has proven inefficacious, giving Ras the reputation of being undruggable (27).

Importantly, interactions between Ras and its upstream and downstream effector proteins occur on the inner leaflet of the cellular membrane. Ras proteins are present in the cell in several variants (HRas, NRas, KRas4a, and KRas4b) that differ primarily in the hypervariable region (HVR), a helical stretch of amino acids extending from the globular catalytic domain that also contains one or more lipophilic tails that insert into the membrane (106). Differences in the HVR serve to target these various Ras proteins into specific regions of the plasma membrane. In KRas4b, the HVR is farnesylated and has a stretch of six lysine residues that interact with anionic residues on the cell membrane (58).

Localization at the membrane surface is essential, not only to increase the effective concentration but also to order the protein correctly such that it can productively interact with its activating and deactivating proteins: Differential positioning of the catalytic domain may promote or prevent binding of upstream or downstream effector proteins. In addition, the precise conformation of Ras, alone and as it interacts with its signaling partners, may differ when membrane bound versus free in solution. Thus, a comprehensive understanding of the biophysics of Ras-membrane interactions may open new opportunities for targeting these pathways. The ease of forming Nanodiscs with precisely defined lipid composition, their inherent stability in solution, and the ability to conjugate the scaffold protein to myriad different reporter groups make Nanodiscs an ideal platform for characterizing Ras-membrane binding.

Recent work in collaboration with investigators at the Frederick National Laboratory for Cancer Research (Maryland, USA) used the Nanodisc system to document the affinity of KRas4b for a membrane surface with defined area and composition. An increase in the affinity of KRas4b for His-tagged Nanodiscs was determined by SPR to be a function of increased anionic lipid concentration (48). However, classic SPR measurements are often complicated by boundary layer diffusion problems (82). Hence, the single-frequency fluorescence anisotropy decay experiment described above was used to provide a label-free means to quantitate this association. These experiments used RuBiPy, a long-lifetime fluorophore, for a solution-based assay to quantitate binding as a function of anionic lipid composition, either DMPS or PIP2 assembled into Nanodiscs (53). These results are reproduced in Figure 3a. Obvious from this analysis is the remarkable similarity to the specificity observed with talin binding, despite being completely different systems and totally different assay methods.

Figure 3.

(a) Binding free energy as KRas4b binds to Nanodiscs prepared with varying DMPS or PIP2 content. Total charge on the Nanodisc is plotted on the abscissa. (b) Binding of KRas4b to Nanodiscs under tight binding conditions (1-μM Nanodiscs). The inflection point at 4-μM KRas4b is consistent with two Ras molecules per leaflet (inset schematic). Abbreviations: DMPS, dimyristoyl phosphatidylserine; PIP2, phosphatidylinositol 4,5-biphosphate; Z_DHH, Debye-Huckle-Henry charge.

Figure 3b demonstrates that Nanodiscs can be used to reveal the formation of a Ras dimer on the membrane surface. Here, KRas4b is added to Nanodiscs in the tight binding regime. Because Nanodiscs in solution provide two equivalent membrane surfaces, a binding stoichiometry of 4:1 suggests a dimer on both faces.

To reveal a possible origin of the apparent preference of KRas4b for PIP2 verses singly charged phospholipids such as DMPS, we conducted long-term molecular dynamics simulations. These studies revealed the presence of long-lived bifurcated salt bridges between PIP2 lipids and basic residues in the HVR and the catalytic domain. Whereas the average residence time for these DMPS/KRas4b contacts was 2.2 ns, those between the protein and PIP2 were 40 ns (53). These differences in residence times satisfactorily explain the specificity of KRas4b for PIP2 observed in vitro and highlight the power of coupling in vitro experiments using Nanodiscs with molecular dynamics simulations.

Nanodiscs can also be readily employed to characterize recruitment of upstream and downstream effector proteins to Ras proteins anchored to a membrane surface. One such application is the documentation of Raf kinase activation by KRas4b. Raf kinase is a principal signaling component of the MAPK pathway and is directly recruited to the plasma membrane from the cytoplasm by KRas4b in its active GTP-bound state (107). DMPS-containing Nanodiscs with a pentahistidine tag were bound to a nickel-chelating donor bead in an Alpha Screen^® assay. Fully processed KRas4b was subsequently bound to Nanodiscs in both active GTP-bound and inactive GDP-bound states. Recruitment of CRaf Ras binding domains conjugated to a glutathione sepharose acceptor bead to the donor complex was then monitored (48). These results clearly revealed that only the GTP-bound KRas4b recruits the CRaf Ras binding domain.

Recently, the Ikura lab (85) employed a versatile combination of Nanodiscs and solution NMR to study the orientation of KRas4b on a Nanodisc surface. In these experiments, isoleucine residues were labeled with ¹³C at the Cδ methyl groups and Ras was tethered to the membrane by conjugation of the protein to a maleimide-functionalized lipid. Nanodiscs were prepared with gadolinium-conjugated lipids, and broadening of isoleucine resonances was observed as a function of the bound nucleotide. These experiments clearly revealed two isoleucine residues that were highly sensitive to activation state: Ile36 near the switch 1 region and isoleucine 139 near helix 4. When GDP is bound to Ras, the protein adopts a conformation in which switch I and switch II regions are solvent exposed, and helix 4 makes contacts with the membrane surface. Paradoxically, when a nonhydrolyzable GTP analog is bound, the protein preferentially occupies an orientation with the C terminus of switch I occluded by the membrane surface such that binding of Raf might be unfavorable. Despite this, binding of the ARaf Ras binding domain to the complex results in KRas4b populating a third orientation, intermediate to the GTP- and GDP-bound states (85).

GENERAL CONCLUSIONS AND FUTURE DIRECTIONS

Nanodiscs have demonstrated great utility in revealing the structure and function of membrane proteins. In this review, we attempt to provide an inroad into the vast literature of Nanodisc technology and contributions to it by laboratories and investigators worldwide. We summarize briefly some of the many biophysical techniques that are now compatible with membrane proteins assembled into Nanodiscs. We describe three different systems in which specificity for a given phospholipid was revealed via distinct assay methods capable of forming a soluble membrane surface of defined area, charge, and lipid composition. With the ready availability of genes for coding various MSPs, purified proteins, and detailed published protocols (40), the number of successful uses will surely grow.