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The structure and properties of gold nanoparticles make them useful for a wide array of biological application. Toxicity, however, has been observed at high concentrations using these systems. MTT, hemolysis, and bacterial viability assays were used to explore differential toxicity among the cell types used, using 2 nm core particles. These studies show that cationic particles are moderately toxic, whereas anionic particles are quite nontoxic. Concentration-dependent lysis mediated by initial electrostatic binding was observed in dye release studies using lipid vesicles, providing the probable mechanism for observed toxicity with the cationic MMPCs.
G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.
Recent structural and biophysical studies point to special roles of anionic lipids in regulating GPCR activity. Biochemical data indicated anionic lipids strongly influenced selectivity of the β2-adrenergic receptor (β2AR) for different G proteins through lipid-protein charge complementarity14, and anionic lipids impacted the affinity and efficacy of β2AR ligands11. Anionic lipids, including POPS, were found to be critical for activation of the CB2 cannabinoid receptor15. Mass spectrometry data supported the role of the anionic phospholipid PtdIns(4,5)P2 (PIP2) stabilizing complexes of human GPCRs with their partner G proteins16. Anionic phospholipids such as PIP2 have also been observed bound to the human serotonin receptor 5-HT1A in cryo-electron microscopy structures17, supporting the idea that direct lipid-protein interactions modulate receptor activity.
Knowledge of GPCR structural plasticity is critical to developing an understanding of signaling mechanisms. Nuclear magnetic resonance (NMR) spectroscopy is well-suited to experimentally investigate and measure GPCR structural plasticity, as it provides the unique capability to detect multiple, simultaneously populated receptor conformations and link observed conformational equilibria to efficacies of bound drugs18,19. We leveraged this capability to investigate the impact of anionic lipids on the structural plasticity of the human A2A adenosine receptor (A2AAR), a representative class A GPCR and exemplary GPCR for investigating receptor-lipid interactions. Our investigation builds on accumulated data from NMR spectroscopic studies of A2AAR interactions with small molecule ligands12,20,21,22,23,24,25,26, providing a firm foundation for evaluating the impact of anionic lipids. In both mass spectrometry experiments16 and molecular dynamics simulations27, anionic lipids were observed to strongly influence the formation of A2AAR signaling complexes. However, a mechanistic basis for these observations has not been determined.
Using nanodiscs, we precisely controlled phospholipid composition to investigate the response of A2AAR over a wide range of defined binary lipid mixtures. In 19F-NMR data, we observed the impact of anionic lipids on the conformational equilibria of A2AAR to be of a similar magnitude as bound drugs. Synergy observed between the presence of anionic lipids and efficacy of bound drugs indicated that sensitivity to lipid composition depended on the receptor conformation. In both NMR experiments and computational models, positively charged residues on the A2AAR intracellular surface near regions that interact with partner signaling proteins appeared to facilitate the influence of anionic lipids. Integrating these data with correlative signaling assays and NMR experiments of judiciously selected A2AAR variants provided a mechanistic view on the role of lipids in A2AAR signaling. Key residues identified in these experiments are conserved among not only class A but all receptor classes, indicating this mechanism may be shared across the GPCR superfamily.
We expressed human A2AAR containing a single extrinsic cysteine introduced into position 289, A2AAR[A289C] (Supplementary Fig. 1). The extrinsic cysteine, located near the intracellular-facing surface of transmembrane (TM) helix VII, was introduced for 19F-NMR experiments, and this A2AAR variant was previously shown to retain pharmacological activity of the native receptor22,28. Purified A2AAR[A289C] was reconstituted into lipid nanodiscs formed with the membrane scaffold protein MSP1D129 containing defined binary mixtures of different molar ratios of zwitterionic phospholipids, POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) or POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), and anionic lipids, POPS (1-palmitoyl- 2-oleoyl-sn-glycero-3-phospho-L-serine), POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) or PI(4,5)P2 (1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-biphosphate)). Analytical characterization of purified lipid nanodiscs containing A2AAR[A289C] showed highly monodispersed and homogenous samples (Supplementary Fig. 1). 31P-NMR in aqueous solutions was used to verify the lipid composition within the nanodisc samples studied in subsequent biophysical and NMR spectroscopy experiments (Supplementary Fig. 2). As the lipid headgroups showed resolved signals with unique chemical shifts, the intensities of the observed 31P signals were used to quantify the relative amounts of each lipid species present. For all samples and for all employed lipids, the relative amounts of each lipid species in the nanodisc samples determined by 31P-NMR closely agreed with the amounts we intended to incorporate in the nanodiscs (Supplementary Fig. 2).
The pharmacological activity of A2AAR[A289C] in lipid nanodiscs containing different binary lipid mixtures was measured in radioligand competition binding assays for both complexes with the antagonist ZM241385 and the agonist NECA. KD values were determined in nanodiscs containing only POPC lipids and mixtures of POPC with one of three different anionic lipids. Measured KD values for the antagonist ZM241385 varied only by a factor of 2 among the different lipid compositions (Supplementary Fig. 5). Measured KI values for the agonist NECA varied only by a factor of 3 among different lipid compositions (Supplementary Fig. 5). These relatively small differences indicated A2AAR[A289C] is pharmacologically active in the range of lipid compositions studied and, the presence of anionic lipids appeared to not impose a significant difference on the pharmacological activity of A2AAR.
To investigate whether observations in Fig. 2 were specific to POPS or applied more generally to additional anionic lipids, we prepared a series of agonist-bound A2AAR samples in nanodiscs containing binary mixtures of POPC and a second, different anionic lipid. 19F-NMR data of agonist-bound A2AAR were all qualitatively highly similar for nanodiscs containing POPC mixed with one type of anionic lipid, including with POPS, POPA, POPG, or PI(4,5)P2 (Fig. 3b). In all mixtures of POPC with anionic lipids, we saw a significant population of the peak P4 corresponding to activated A2AAR (Fig. 3b). To also investigate the possibility that POPC played a special role in the binary mixtures, we recorded NMR data with agonist-bound A2AAR in nanodiscs containing POPE mixed with POPS. NMR data with these samples was highly similar to samples prepared with POPC and POPS, showing the presence of activated A2AAR with a population that increased proportionally with increasing amounts of anionic lipids (Supplementary Fig. 10). These data support that activation of agonist-bound A2AAR could be achieved in the presence of any anionic lipid studied regardless of the phospholipid headgroup chemical structure. We also determined the integrals of each peak from the deconvolutions of all the spectra and tabulated these values as a fraction of the total integrated signal intensities (Supplementary Table 1). For all spectra shown in Fig. 3b we observed comparable integrated intensities for states, with the state P4 showing the largest fraction in the presence of PIP2 (Supplementary Table 1). We also observed larger line widths for states P4 and P2 for the A2AAR agonist complex in nanodiscs containing mixtures of POPC and POPA, suggesting the active state exhibits a larger degree of structural plasticity for this lipid composition.
a A 1D 19F-NMR spectrum of the antagonist-A2AAR[A289CTET] complex in lipid nanodiscs containing a mixture of POPC and POPS lipids. b The 1D 19F NMR spectra of an agonist-A2AAR[A289CTET] complex in lipid nanodiscs containing either only POPC lipids or a mixture of POPC and anionic lipids, as indicated on the right of each spectrum. The chemical structures of the lipid headgroups are shown with charges expected at physiological pH.
Our 19F data showed anionic lipids impacted the conformational ensemble of agonist-bound but not antagonist-bound A2AAR. We therefore hypothesized positively charged residues located near the intracellular surface that show significant differences in conformation between active and inactive states could play important roles mediating the impact of anionic lipids. We identified 4 positively charged residues predicted by the Orientation of Proteins in Membranes (OPM) database35 to be near the lipid-bilayer boundary: R1995.60, H2306.32, K2336.35 and R2917.56 (superscripts denote the Ballesteros-Weinstein nomenclature36) and in different conformations between agonist-bound and antagonist-bound A2AAR crystal structures (Fig. 4a). Though our experiments were carried out at neutral pH, H2306.32 has been proposed to be positively charged37 and was thus included in the current study. Positively charged amino acids are frequently found in position 6.32 in other GPCRs (see Discussion). 59ce067264