Escaping the flatlands: new approaches for studying the dynamic assembly and activation of GPCR signaling complexes

Abstract

Despite significant recent advances in molecular and structural studies of G protein-coupled receptors (GPCRs), an understanding of transmembrane signal transduction with chemical precision requires new approaches. Simple binary receptor-ligand or receptor-G protein complex models cannot adequately describe the relevant macromolecular signaling machineries. GPCR signalosomes undergo complex dynamic assembly-disassembly reactions to create allosteric signaling conduits whose properties cannot necessarily be predicted from individual elements alone. The combinatorial possibilities inherent in a system with hundreds of potential components suggest that high-content miniaturized experimental platforms and computational approaches will be required. To study allosteric effects involved in signalosome reaction pathways, a bottom-up approach using multicolor single-molecule detection fluorescence experiments in biochemically defined systems and complemented by molecular dynamics models of macromolecular complexes is proposed. In bridging the gap between molecular and systems biology, this synthetic approach suggests a way forward from the flatlands to multi-dimensional data collection.

Figure 1. Detection and amplification of signals: dynamic GPCR signalosomes as a macromolecular machine

(a) Cyclic sequence (activation, desensitization, and recycling) of the key states in the GPCR signal transduction process. The corresponding characteristic macromolecular complexes or signalosomes are receptor–G protein (RG), receptor–GPCR kinase (RGRK), phosphorylated receptor–arrestin (R^PArr), and phosphorylated receptor–protein phosphatase (R^PPP). Note the quasi-irreversible reaction resulting in de-phosphorylated (R) and phosphorylated receptor (R^P), respectively. (b) Molecular model of a representative GPCR signalosome, the receptor–G protein complex. Secondary structure cartoon (left) and space-filling model (right) illustrate the receptor–G protein complex in a phospholipid bilayer.

Figure 2. Functional reconstitution of CCR5 in solid-supported tethered phospholipid bilayer membranes

We developed the following method to self-assemble the depicted structure by sequential perfusion of the different protein solutions in a simple semi-microfluidics chamber [93]. The glass surface is first coated with octadecyl (C18) trialkoxy-silane. The binding capacity of the C18 modified glass for biotinylated BSA is substantially higher as compared to untreated glass [40]. The adsorbed layer of biotinylated BSA is further stabilized by neutravidin (A), a deglycosylated form of the tetrameric biotin-binding protein avidin with a more neutral isoelectric point. The anti-rhodopsin 1D4 mAb that recognizes the C-terminal nonapeptide of rhodopsin (“C9-tag”) is biotinylated specifically on the Fc stem. It is captured by avidin and the remaining biotin binding sites are saturated with biotin-PEG2000-DSPE. The mAb captures heterologously expressed CCR5 with an engineered C-terminal C9-tag from detergent solution. A lipid bilayer (light green) is formed by dilution of POPC/CHAPS bicelles. The fluorescently labeled chemokine (CCL3/MIP-1α-Alexa-647) is added in the last step, utilizing BSA as carriers.

Figure 3. Representative SMD-TIRF images of single chemokine molecules bound to CCR5 in tethered membranes

(a) Image obtained with the SMD-TIRF microscope after subtraction of static background from ‘hot’ pixels of the CCD sensor and after application of a 2D convolution with a spot enhancing filter that was shown to be the optimal detector of a Gaussian-like spot in 1/ω² noise [95]. It shows two classes of single-fluorophore spots. One side of the quadratic image corresponds to 40 μm in the sample plane. (b) Time series of the indicated region-of-interest from (a) with 10 seconds exposure time taken in one-minute intervals. The bright central spot corresponds to single fluorescently-labeled chemokines (CCL3/MIP-1α-Alexa-647) bound to CCR5 embedded in a bilayer membrane (see Figure 3) that is visible from 115 to 126 min. (c) Raw image corresponding to (a) before image processing. (d) A surface plot illustrating intensity of the spots as peaks. The tall signals are from single ligand-receptor complexes. The small signals (about 15% of the height of the Alexa-647 signals) are due to hydrophobic fluorescent impurities in a buffer to improve photostability and blinking of Alexa-647 [96]. We found that these impurities can efficiently be removed (not shown) by hydrophobic-affinity chromatography of the employed enzymes (glucose oxidase or catalase). This scheme allows acquisition of hundreds of images with sufficient signal-to-noise ratio to identify individual receptor-bound chemokine molecules before irreversible photobleaching.

Figure I

Examples of allosteric modulators (A) are pharmacological agents (agonists, partial agonists, inverse agonists, neutral antagonists, biased ligands), dimerizing receptors, chemical receptor modifications (side-chain protonation or phosphorylation), and physicochemical environmental effects (membrane curvature stress, lipid composition). Despite significant progress in the structural biology of G proteins, little is known about the precise interaction of a G protein with a GPCR (or GPCR dimer), and how this interaction leads to GDP release and the subsequent GTP uptake by the active complex [5]. Intermediate states of the receptor- and Gβγ-dependent nucleotide exchange process and GTPase cycle of Gα are shown. Note that for each state, multiple conformational substates of the participating proteins are possible. These conformational substates might be rapidly interconverting, or alternatively, they might correspond to metastable long-lived conformations that exhibit hysteretic behavior (conformational memory). A pharmacological agonist ligand functions as an allosteric modulator (A) that changes the distribution of conformational substates. The corresponding perturbation of the receptor free energy landscape will affect the binding of the inactive heterotrimer, Gα[GDP]Gβγ, and destabilization of its bound GDP. Structures for components of the lower part of the cycle have been reported.