The importance of ligands for G protein-coupled receptor stability

doi:10.1016/j.tibs.2014.12.005

Trends in Biochemical Sciences

Volume 40, Issue 2, February 2015, Pages 79-87

https://doi.org/10.1016/j.tibs.2014.12.005 Get rights and content

Highlights

•
Ligands stabilize GPCRs and affect their function.
•
A case study on the A_2A adenosine receptor and ligand influences.
•
Ligand-induced receptor stabilization plays role in successful GPCR crystallography.

Traditionally, G protein-coupled receptor (GPCR) activity has been characterized by ligand properties including affinity (K_i), potency (IC₅₀/EC₅₀), efficacy (E_max), and kinetics (K_on/K_off). These properties are related to ligand residence time, a general index of drug–target interaction in vivo. Recent GPCR structure–function breakthroughs have all required ligand stabilization of the receptor in some manner, highlighting the natural instability of these important cell surface receptors. This research has initiated a new era of discovery that highlights the importance of ligand–receptor interactions beyond the traditional mindset. We propose that receptor stability is related to receptor folding and residence in the cell membrane, affording a new dimension that should be considered when studying receptor function.

Section snippets

Overview of GPCRs and ligands

GPCRs constitute the largest protein superfamily. GPCRs have more than 800 members in the human genome, and these can be divided into: rhodopsin-like (class A), secretin-like and adhesion (class B), glutamate (class C), and frizzled/Tas2 (class F) subfamilies. They share a conserved seven-transmembrane (7TM) α-helical bundle structure. Because of their diverse physiological and pathological roles, GPCRs are the targets for approximately 30–50% of drugs currently on the market or in clinical

Influence of ligands on receptor stability

To date, all the GPCR structures that have been determined have had a ligand bound, either in the orthosteric or allosteric binding sites (Figure 1). The reason for this is that the presence of a ligand plays an important role in GPCR stabilization. Ligands recognize and interact with a GPCR binding site, which can promote folding of the correct conformation and lock the receptor in a stable state. Furthermore, in our work to improve surface expression and facilitate stabilization of GPCRs, we

Influence of receptor variants

In addition to ligand stabilization, receptor variants also have a large influence on receptor stability (Table S1 in the supplementary material online) and crystallization. GPCR construct design is usually considered to be a useful approach to altering GPCR sequence to improve receptor stability, including truncation of receptor N- or C-termini; the addition of a soluble fusion partner; point stabilizing mutations; and systematic alanine (or leucine) scanning. We describe these approaches,

Concluding remarks

The key to successful stabilization of GPCRs is, in many cases, to find the best construct and ligand combination that can form a stable receptor–ligand complex. To improve receptor stability and functional conformation, we introduce fusion proteins, truncations, or mutations; we screen ligands or antibodies; we optimize expression and purification conditions; and we characterize construct and ligand function. We suggest that the second part of the equation is to search for ideal ligands for

Acknowledgments

The authors acknowledge ShanghaiTech University and Shanghai Municipal Government for financial support; and Angela Walker, Kate White, Michael Hanson, and Gaojie Song for careful review of this manuscript.

References (59)

E. Chun
Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors
Structure
(2012)
A.I. Alexandrov
Microscale fluorescent thermal stability assay for membrane proteins
Structure
(2008)
V. Katritch
Diversity and modularity of G protein-coupled receptor structures
Trends Pharmacol. Sci.
(2012)
C.B. Roth
Stabilization of the human beta2-adrenergic receptor TM4-TM3–TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure
J. Mol. Biol.
(2008)
A.S. Dore
Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine
Structure
(2011)
I. Kufareva
Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment: meeting new challenges
Structure
(2014)
W. Liu
LCP-Tm: an assay to measure and understand stability of membrane proteins in a membrane environment
Biophys J.
(2010)
V. Katritch
Allosteric sodium in class A GPCR signaling
Trends Biochem. Sci.
(2014)
J.A. Gray et al.
Paradoxical trafficking and regulation of 5-HT(2A) receptors by agonists and antagonists
Brain Res. Bull.
(2001)
T. Kenakin et al.
Defining and characterizing drug/compound function
Biochem. Pharmacol.
(2014)

A. Manglik

Crystal structure of the mu-opioid receptor bound to a morphinan antagonist

Nature

(2012)

Cited by (64)

Magic angle spinning NMR of G protein-coupled receptors
2022, Progress in Nuclear Magnetic Resonance Spectroscopy
G protein-coupled receptors (GPCRs) have a simple seven transmembrane helix architecture which has evolved to recognize a diverse number of chemical signals. The more than 800 GPCRs encoded in the human genome function as receptors for vision, smell and taste, and mediate key physiological processes. Consequently, these receptors are a major target for pharmaceuticals. Protein crystallography and electron cryo-microscopy have provided high resolution structures of many GPCRs in both active and inactive conformations. However, these structures have not sparked a surge in rational drug design, in part because GPCRs are inherently dynamic and the structural changes induced by ligand or drug binding to stabilize inactive or active conformations are often subtle rearrangements in packing or hydrogen-bonding interactions. NMR spectroscopy provides a sensitive probe of local structure and dynamics at specific sites within these receptors as well as global changes in receptor structure and dynamics. These methods can also capture intermediate states and conformations with low populations that provide insights into the activation pathways. We review the use of solid-state magic angle spinning NMR to address the structure and activation mechanisms of GPCRs. The focus is on the large and diverse class A family of receptors. We highlight three specific class A GPCRs in order to illustrate how solid-state, as well as solution-state, NMR spectroscopy can answer questions in the field involving how different GPCR classes and subfamilies are activated by their associated ligands, and how small molecule drugs can modulate GPCR activation.
Optimizing the α<inf>1B</inf>-adrenergic receptor for solution NMR studies
2020, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Therein, thermal unfolding is monitored by the increase in fluorescence of the CPM dye upon binding to cysteines and hydrophobic patches exposed during protein denaturation [18]. Fig. 4D shows that the α1B-AR-B1 receptor construct has an apparent Tm of 76 °C in DDM/CHS, indicating an exceptionally high stability compared to other GPCRs measured under similar conditions [19]. The Kd values for the wild-type (wt) α1B-AR receptor as well as for the B1 and #12 mutants were determined using a RLBA.
Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes ¹³C, ¹⁵N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the α_1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1–2 mg/L of D₂O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the α_1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [¹⁵N,¹H]- as well as [¹³C,¹H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [¹³C,¹H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.
Structural biology of human GPCR drugs and endogenous ligands - insights from NMR spectroscopy
2020, Methods
Citation Excerpt :
GPCR crystal structures and, more recently, cryoEM structures have provided the vast majority of information on receptor-ligand interactions. Crystallization of drug-receptor complexes is generally facilitated by the use of ligands with relatively strong affinities (e.g. KD < 100 nM) and specific molecular properties [19–21], which can limit the potential chemical space of GPCR drugs for which structural information can be obtained by this method alone. Complementing this approach, NMR spectroscopy in aqueous solutions and solids can provide structural data of bound drugs for compounds with both strong and weak affinities (KD > 100 nM), higher off-rates, or for ligands that exhibit dynamic behavior even while bound to the receptor [18].
G protein-coupled receptors (GPCRs) represent the largest class of “druggable” proteins in the human genome. For more than a decade, crystal structures and, more recently, cryoEM structures of GPCR complexes have provided unprecedented insight into GPCR drug binding and cell signaling. Nevertheless, structure determination of receptors in complexes with weakly binding molecules or complex polypeptides remains especially challenging, including for hormones, many of which have so far eluded researchers. Nuclear magnetic resonance (NMR) spectroscopy has emerged as a promising approach to determine structures of ligands bound to their receptors and to provide insights into the dynamics of GPCR-bound drugs. The capability to investigate compounds with weak binding affinities has also been leveraged in NMR applications to identify novel lead compounds in drug screening campaigns. We review recent structural biology studies of GPCR ligands by NMR, highlighting new methodologies enabling studies of GPCRs with native sequences and in native-like membrane environments that provide insights into important drugs and endogenous ligands.
Production of membrane proteins in industry: The example of GPCRs
2020, Protein Expression and Purification
Citation Excerpt :
A ligand inducing a high melting temperature (Tm) value implies that the receptor has formed a stable complex with the bound ligand and in turn generated a stable conformational state. Analysis by Zhang et al. suggested that a Tm value higher than 55 °C identified through a CPM assay system is usually required, although not sufficient, for successful crystallisation and subsequent crystal diffraction [84]. Physical chemical properties of ligands also need to be considered; identification of ligands that are soluble and remain in solution at the high concentrations >1000 fold over the KD to provide complete occupancy of the ligand-binding site is property to consider.
Whereas membrane proteins make up ∼23% of the human proteome, it is estimated that membrane proteins constitute more than 60% of current drug targets. With membrane proteins forming such a high percentage of drug targets relative to their abundance within the proteome, it is little wonder that drug companies need to rapidly access high quality membrane proteins for their drug discovery process. Newly devised technologies, such as rapid gene synthesis, novel detergents, and protein thermostabilisation strategies allow conventionally ‘undruggable’ membrane proteins to be drugged. In this review, we survey the state-of-the-art gene design, expression and purification strategies, and protein thermostabilisation methods used within a modern drug discovery programme, with a focus on G protein-coupled receptors.
Impact of GPCR Structures on Drug Discovery
2020, Cell
Structures of 70 unique G protein-coupled receptors (GPCRs) have been determined, with over 370 structures in total bound to different ligands and the receptors in various conformational states. Structure-based drug design has been applied to an increasing number of GPCR targets over the past decade and now a few of these drug candidates have entered clinical trials. Given the length of time required for a drug to reach the market, there are no documented examples of licensed drugs being developed with the aid of a structure, but this is likely to change as current efforts come to fruition.
Structure-based discovery of novel inhibitors of Mycobacterium tuberculosis CYP121 from Indonesian natural products
2020, Computational Biology and Chemistry
Tuberculosis (TB) continues to be a serious global health threat with the emergence of multidrug-resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). There is an urgent need to discover new drugs to deal with the advent of drug-resistant TB variants. This study aims to find new M. tuberculosis CYP121 inhibitors by the screening of Indonesian natural products using the principle of structure-based drug design and discovery. In this work, eight natural compounds isolated from Rhoeo spathacea and Pluchea indica were selected based on their antimycobacterial activity. Derivatives compound were virtually designed from these natural molecules to improve the interaction of ligands with CYP121. Virtual screening of ligands was carried out using AutoDock Vina followed by 50 ns molecular dynamics simulation using YASARA to study the inhibition mechanism of the ligands. Two ligands, i.e., kaempferol (KAE) and its benzyl derivative (KAE3), are identified as the best CYP121 inhibitors based on their binding affinities and adherence to the Lipinski’s rule. Results of molecular dynamics simulation indicate that KAE and KAE3 possess a unique inhibitory mechanism against CYP121 that is different from GGJ (control ligand). The control ligand alters the overall dynamics of the receptor, which is indicated by changes in residue flexibility away from CYP121 binding site. Meanwhile, the dynamic changes caused by the binding of KAE and KAE3 are isolated around the binding site of CYP121. These ligands can be developed for further potential biological activities.

View all citing articles on Scopus

View full text

Trends in Biochemical Sciences

OpinionThe importance of ligands for G protein-coupled receptor stability

Highlights

Section snippets

Overview of GPCRs and ligands

Influence of ligands on receptor stability

Influence of receptor variants

Concluding remarks

Acknowledgments

Structure

Structure

Trends Pharmacol. Sci.

J. Mol. Biol.

Structure

Structure

Biophys J.

Trends Biochem. Sci.

Brain Res. Bull.

Biochem. Pharmacol.

Methods Neurosci.

Structure

Structure

Neuropharmacology

Drug design strategies for targeting G-protein-coupled receptors

Chembiochem

Ice breaking in GPCR structural biology

Acta Pharmacol. Sin.

Structure–function of the G protein-coupled receptor superfamily

Annu. Rev. Pharmacol. Toxicol.

Crystal structure of rhodopsin: a G protein-coupled receptor

Science

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Science

Structure of a beta1-adrenergic G-protein-coupled receptor

Nature

Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists

Science

Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex

Science

The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist

Science

Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist

Science

Structure of the human histamine H1 receptor complex with doxepin

Nature

Crystal structure of a lipid G protein-coupled receptor

Science

Structure and dynamics of the M3 muscarinic acetylcholine receptor

Nature

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

Nature

Crystal structure of the mu-opioid receptor bound to a morphinan antagonist

Nature

Opinion
The importance of ligands for G protein-coupled receptor stability