Chapter Four - Prediction and targeting of GPCR oligomer interfaces

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Abstract

GPCR oligomerization has emerged as a hot topic in the GPCR field in the last years. Receptors that are part of these oligomers can influence each other's function, although it is not yet entirely understood how these interactions work. The existence of such a highly complex network of interactions between GPCRs generates the possibility of alternative targets for new therapeutic approaches.

However, challenges still exist in the characterization of these complexes, especially at the interface level. Different experimental approaches, such as FRET or BRET, are usually combined to study GPCR oligomer interactions. Computational methods have been applied as a useful tool for retrieving information from GPCR sequences and the few X-ray-resolved oligomeric structures that are accessible, as well as for predicting new and trustworthy GPCR oligomeric interfaces.

Machine-learning (ML) approaches have recently helped with some hindrances of other methods. By joining and evaluating multiple structure-, sequence- and co-evolution-based features on the same algorithm, it is possible to dilute the issues of particular structures and residues that arise from the experimental methodology into all-encompassing algorithms capable of accurately predict GPCR-GPCR interfaces.

All these methods used as a single or a combined approach provide useful information about GPCR oligomerization and its role in GPCR function and dynamics. Altogether, we present experimental, computational and machine-learning methods used to study oligomers interfaces, as well as strategies that have been used to target these dynamic complexes.

Introduction

G protein-coupled receptors (GPCRs) superfamily has been a subject of high interest in cell and molecular biology field for decades, mainly due to its presence in various physiological events. However, their mechanism of action is not yet fully understood. This family also represents 34% of the drugs approved by the Food and Drug Administration (FDA), demonstrating its high viability as therapeutic targets.1 GPCRs are highly dynamical proteins that mediate the signal transduction triggered by extracellular stimuli through the cell membrane. The family has up to 800 different receptors divided by their structural and functional similarities into 5 major subfamilies: class A, B, C, frizzled, and adhesion. GPCRs have a common structure present through the different subfamilies: seven transmembrane domains (TM), connected by three extracellular (ECL) and three intracellular loops (ICL), with N-terminal in the extracellular side and the C-terminal on the intracellular side. The TM region is highly conserved, and interhelical bonds and hydrophobic interactions maintain its stability. Loops are the least conserved regions and display structural variability between the subfamilies.2, 3

For many years the GPCR family members have been studied as monomeric entities; however, in recent years, accumulating evidence has shown that GPCRs can function in dimeric (homo and hetero) or higher-order oligomeric states. Class C GPCRs are known to form dimers constitutively through their extensive extracellular domain to work.4 Class A has increasingly data pointing toward the existence of homo and heterodimers.5, 6 The ratio between monomeric and dimeric states is a defining characteristic of this subfamily. There is evidence that suggests that in receptors like Β2 adrenergic receptor (B2AR) and α1B-adrenergic receptor (α1BAR), dimerization is necessary for efficient surface localization.7, 8 Thus, some authors suggested that dimers are assembled during biosynthesis, perhaps inside the endoplasmic reticulum.9, 10, 11 Nevertheless, recent data proposes a dynamical view of GPCR dimers that are in equilibrium with their monomeric forms and have variable timescales, depending on the membrane or cellular environments (such as cytoskeleton and scaffolding or anchoring proteins).9, 12, 13, 14, 15

The effects of dimerization/oligomerization on the structure and dynamics of receptors are not yet entirely understood, neither their implication in human physiology and pathology. However, the steady increase in studies related to the allosteric interactions between the receptors in complex have brought light into this subject. A simple way to view the importance of these interactions is to categorize them in three groups as it was done by Guidolin et al.16: (a) neighbor receptors can modulate each other's orthosteric binding site; (b) receptors can modulate the intracellular binding pocket, thus altering signaling pathways; (c) or new allosteric sites can emerge for binding with different modulators.

There are currently several curated and specialized databases where information concerning 3D structures of GPCRs can be found, including dimers, and other membrane proteins (MPs). Some of these databases include: (a) the MPs of known 3D structure (mpstruc)17 that identifies and collect MPs of the PDB data bank (as of October 7, 2019 it contains 952 unique entries); (b) the Transporter Classification DataBase (TCDB)18 that provides functional and phylogenetic information on membrane transport proteins (as of October 7, 2019 it contains ~ 1405 families of transport proteins); (c) the Protein Data Bank of Transmembrane Proteins (PDBTM)19 that uses TMDET algorithm20 in all PDB entries for location of TM protein in the lipid bilayer (as of October 7, 2019 it contains 4084 transmembrane proteins); (d) the Orientations of Proteins in Membrane (OPM) database21 that uses PPM server to provide spatial arrangements of MPs with respect to the hydrocarbon core of the lipid bilayer; (e) the MemProtMD, a meta-database that presents the results of molecular dynamics simulations of some MPs of mpstruc embedded in lipid bilayers (the database contains ~ 3500 intrinsic MPs structures)22; and (f) more specific databases for GPCRs such as the G-Protein Coupled Receptor Database (GPCRdb) with 15,147 proteins (as of October 7, 2019),23 the G-Protein Coupled Receptor Oligomerization Knowledge Base (GPCR-OKB),24 or the GPCR-HGmod25 that contains 1026 putative 3D structural models of GPCRs in the human genome generated by the GPCR-I-TASSER pipeline and deposited in the GPCR-EXP (database of experimentally solved and predicted GPCR structures) (https://zhanglab.ccmb.med.umich.edu/GPCR-EXP/). Known GPCR-GPCR interactions are stored and can be acquired through GPCR-OKB24 and GPCR-HetNet.26

Currently, there are 12 structures of GPCR dimers in PDB that present a crystallographic asymmetric unit and with a software-determined quaternary structure27 (PDB id: 2VT4,28 4GPO,29 3ODU,30 3OE9,30 4EA3,31 6AK3,32 5O9H,33 5ZKQ,34 3CAP,35 2PED,36 2J4Y,37 4JKV,38 6N5239). Furthermore, three additional structures are found as an asymmetric unit but with no quaternary structure prediction: two from class A (PDB id: 5UEN,40 4DJH41) and one from class C (PDB id: 2E4U42).

The existence of this highly complex network of interactions between GPCRs and how they can modulate each other's behavior contributed to the development of new therapeutic approaches. Nevertheless, the challenges in characterizing these complexes remain, and in particular at the interface level, which plays a unique role in the development of new targeting drugs. Herein, we present experimental, in silico computational methods and ML methods that are currently in use for the characterization and interpretation of these interfaces. We also review which strategies have been used to target these dynamic complexes. Some essential key concepts for further understanding of the chapter are presented in Box 1.

Section snippets

Experimental approaches

Experimental-based methods can be applied to study protein-protein interactions (PPIs), including GPCR oligomers. These approaches can be split into four categories, affinity-based methods, proteomics-based methods, fluorescence-based assays and genetic assays. Schiedel et al.43 performed an extensive review about the application of experimental methods to investigate GPCR oligomers. To study PPIs in GPCR oligomerization, different experimental approaches are usually combined, being the most

Targetting PPIs: Orthosteric and allosteric modulation

Targeting PPIs has become a promising strategy in drug discovery since they display a key role in both several biological processes and pathological conditions. Nevertheless, the large and flat interfaces of PPIs make this achievement a challenging task, mostly due to the lack of drug-binding pockets.217, 218

Two main approaches can be applied for targeting PPIs: orthosteric and allosteric modulation, both by using small-molecules or peptidomimetic agents.218, 219 While orthosteric PPI

Concluding remarks

GPCRs are a broad family of membrane receptors that have an essential role in multiple diseases, and because of that are targets of about 34% of total approved drugs. Diverse experimental and computational approaches have demonstrated the existence of GPCR dimers and high-order oligomers and their impact on GPCR function and dynamics. The discovery of GPCR dimers with a physiological importance suggests that new and more targeted drugs can be developed by targeting these structures.

In this

Acknowledgments

I.S.M. was funded by the Fundação para a Ciência e a Tecnologia (FCT) Investigator programme—IF/00578/2014 (co-financed by European Social Fund and Programa Operacional Potencial Humano). This research was funded by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000008: BrainHealth 2020, and through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation and Portuguese national

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