Nanobody stabilization of G protein-coupled receptor conformational states

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Remarkable progress has been made in the field of G protein-coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis.

Highlights

► Last four years, several inactive-state GPCR structures have been solved. ► Active-state structures may be unstable without a native signaling partner. ► Nanobodies act as surrogates of GPCR signaling partners. ► Nanobody 80 has G protein-like properties and stabilizes an agonist activated state of the β2AR.

Introduction

G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and are the most commonly targeted membrane protein class for medicinal therapeutics. Over the past three decades, great progress has been made in characterizing the pharmacology, cellular physiology and in vivo function of many members of this family. The paradigm of GPCR signaling involves activation of heterotrimeric G proteins (Gαβγ). The inactive Gαβγ heterotrimer is composed of two principal elements, Gα•GDP and the Gβγ heterodimer. Gβγ sequesters the switch II element on Gα such that it is unable to interact with other proteins in the second messenger systems. Activated GPCRs catalyze the release of GDP from Gα, allowing GTP to bind and liberate the activated Gα•GTP subunit. In this state, switch II forms a helix stabilized by the γ-phosphate of GTP allowing it to interact with effectors such as adenylyl cyclase. Although much progress has been made in understanding how Gα subunits interact with and regulate the activity of their downstream targets, it is not clear how activated GPCRs initiate this process by catalyzing nucleotide exchange on Gαβγ [1].

In the classical models, signaling by the activated GPCR is terminated by phosphorylation of the cytoplasmatic loops and/or tail of the receptor by GPCR kinases. This results in the binding of arrestins that mediate receptor desensitization and internalization via clathrin-coated pits. This classical model is both oversimplified and incomplete. Over the past decade, we learned that arrestins act not only as regulators of GPCR desensitization but also as multifunctional adaptor proteins that have the ability to signal through multiple effectors such as MAPKs, SRC, NF-kB, and PI3K [2]. In this revised model, β-arrestins are interacting with and recruiting intracellular signaling molecules, as well as mediating desensitization. It is still unclear whether the same receptor conformations that result in arrestin-mediated signal transduction also lead to receptor desensitization. For a number of different receptor systems, it has been found that the G protein dependent and the arrestin dependent signaling events are pharmacologically separable [3]. In other words, a class of ligands referred to as biased agonists selectively trigger signaling toward one pathway over the other; that is, they preferentially signal through either the G protein-mediated or arrestin-mediated pathway [4]. It thus appears that GPCRs, despite their small size, are sophisticated allosteric machines with multiple signaling outputs. Characterizing these functionally distinct structures is challenging, but essential for understanding the mechanism of physiologic signaling and for developing more effective drugs.

Section snippets

Active-state GPCR structures

Polytopic membrane proteins such as GPCRs, transporters and channels are dynamic proteins that exist in an ensemble of functionally distinct conformational states [5]. Crystallogenesis typically traps the most stable low energy states, making it difficult to obtain high-resolution structures of other less stable but biologically relevant functional states. The first structures of rhodopsin covalently bound to 11-cis-retinal represent a completely inactive state with virtually no basal activity [

Nanobodies as G protein surrogates

For GPCRs that do not tolerate acidic conditions, stabilization of an active conformation can be achieved in different ways. The most physiologic approach is to use a native signaling partner such as a G protein or arrestin. Unfortunately, interactions of GPCRs with G proteins or arrestins are highly sensitive to pH, detergents, and nucleotides used during the solubilization and purification of these proteins. It has therefore been difficult to form complexes of sufficient stability for

Nanobody-assisted crystallography of GPCRs

The β2AR•T4L•Nb80 complex was crystallized in lipidic cubic phase [47]. Diffraction data were collected using minibeam technology [48] and the solution was determined by molecular replacement [45••]. Figure 1(a) shows the crystallographic packing of the β2β2AR•T4L•Nb80 complex. Crystallographic contacts are primarily mediated by Nb80. As shown in Figure 1(b), the long CDR3 loop of the nanobody projects into the transmembrane core occupying a position nearly identical to the transducin peptide

Conflicts of interest

The authors declare that they have no conflict of interest.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The work in JS's laboratory is supported by Vrije Universiteit Brussel (VUB, GOA65), Vlaams Instituut Biotechnologie (VIB), the Fund for Scientific Research of Flanders (FWOAL551), the Hercules Foundation (HERC2) and the Institute for the encouragement of Scientific Research and Innovation of Brussels (ISRIB). BKK receives support from National Institutes of Health Grants NS028471 and GM083118 and the Mathers Foundation.

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