Computational Predictions of G Protein-Coupled Receptor Structures and Binding Sites

G protein-coupled receptors (GPCRs) are the largest family of proteins within the human genome. They consist of seven transmembrane (TM) helices, with a N-terminal region of varying length and structure on the extracellular side, and a C-terminus on the intracellular side. GPCRs are involved in transmitting extracellular signals to cells, and as such are crucial drug targets. Designing pharmaceuticals to target GPCRs is greatly aided by full-atom structural information of the proteins. In particular, the TM region of GPCRs is where small molecule ligands (much more bioavailable than peptide ligands) typically bind to the receptors. In recent years nearly thirty distinct GPCR TM regions have been crystallized. However, there are more than 1,000 GPCRs, leaving the vast majority of GPCRs with limited structural information. Additionally, GPCRs are known to exist in a myriad of conformational states in the body, rendering the static x-ray crystal structures an incomplete reflection of GPCR structures. In order to obtain an ensemble of GPCR structures, we have developed the GEnSeMBLE procedure to rapidly sample a large number of variations of GPCR helix rotations and tilts. The lowest energy GEnSeMBLE structures are then docked to small molecule ligands and optimized. The GPCR family consists of five subfamilies with little to no sequence homology between them: class A, B1, B2, C, and Frizzled/Taste2. Almost all of the GPCR crystal structures have been of class A GPCRs, and much is known about their conserved interactions and binding sites. In this work we particularly focus on class B1 GPCRs, and aim to understand that family’s interactions and binding sites both to small molecules and their native peptide ligands. Specifically, we predict the full atom structure and peptide binding site of the glucagon-like peptide receptor and the TM region and small molecule binding sites for eight other class B1 GPCRs: CALRL, CRFR1, GIPR, GLR, PACR, PTH1R, VIPR1, and VIPR2. Our class B1 work reveals multiple conserved interactions across the B1 subfamily as well as a consistent small molecule binding site centrally located in the TM bundle. Both the interactions and the binding sites are distinct from those seen in the more well-characterized class A GPCRs, and as such our work provides a strong starting point for drug design targeting class B1 proteins. We also predict the full structure of CXCR4 bound to a small molecule, a class A GPCR that was not closely related to any of the class A GPCRs at the time of the work.

Chemical-scale studies of G protein-coupled receptors and ligand-gated ion channels

This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.

Chapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural α-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.

In Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased β-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.

Chapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and α4β4 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the α4β4 receptor.

Chapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the α7 nAChR. Additionally, binding interactions to the complementary face of the α7 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.

Chapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.

Chapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.

Structure Prediction of G-Protein Coupled Receptors

G-protein coupled receptors (GPCRs) form a large family of proteins and are very important drug targets. They are membrane proteins, which makes computational prediction of their structure challenging. Homology modeling is further complicated by low sequence similarly of the GPCR superfamily.

In this dissertation, we analyze the conserved inter-helical contacts of recently solved crystal structures, and we develop a unified sequence-structural alignment of the GPCR superfamily. We use this method to align 817 human GPCRs, 399 of which are nonolfactory. This alignment can be used to generate high quality homology models for the 817 GPCRs.

To refine the provided GPCR homology models we developed the Trihelix sampling method. We use a multi-scale approach to simplify the problem by treating the transmembrane helices as rigid bodies. In contrast to Monte Carlo structure prediction methods, the Trihelix method does a complete local sampling using discretized coordinates for the transmembrane helices. We validate the method on existing structures and apply it to predict the structure of the lactate receptor, HCAR1. For this receptor, we also build extracellular loops by taking into account constraints from three disulfide bonds. Docking of lactate and 3,5-dihydroxybenzoic acid shows likely involvement of three Arg residues on different transmembrane helices in binding a single ligand molecule.

Protein structure prediction relies on accurate force fields. We next present an effort to improve the quality of charge assignment for large atomic models. In particular, we introduce the formalism of the polarizable charge equilibration scheme (PQEQ) and we describe its implementation in the molecular simulation package Lammps. PQEQ allows fast on the fly charge assignment even for reactive force fields.

Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Functional evaluation of noncovalent interactions in neuroreceptors and progress toward the expansion of unnatural amino acid methodology

This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.

In chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-π interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-π interaction with dopamine.

Chapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.

Chapter 4 identifies a cation-π interaction between glutamate and a tyrosine residue on loop C in the GluClβ receptor. Using the recently published crystal structure of the homologous GluClα receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.

Chapter 5 examines inherent properties of the GluClα receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.