Exploring G-Protein Coupled Receptor Activation

Exploring G-Protein Coupled Receptor Activation with Multiscale Molecular Simulations

THÈSE NO 5005 (2011) PRÉSENTÉE le 25 mars 2011 À LA FACULTÉ SCIENCES DE BASE

LABORATOIRE DE CHIMIE ET BIOCHIMIE COMPUTATIONNELLES PROGRAMME DOCTORAL EN CHIMIE ET GÉNIE CHIMIQUE

ÉCOLE POLYTECHNIQUE FÉDÉRALE DE LAUSANNE POUR L'OBTENTION DU GRADE DE DOCTEUR ÈS SCIENCES

PAR

Stefano Vanni

acceptée sur proposition du jury: Prof. L. Helm, président du jury Prof. U. Röthlisberger, directrice de thèse Prof. P. Carloni, rapporteur Prof. M. Sansom, rapporteur Prof. H. Vogel, rapporteur

Suisse 2011

Abstract This thesis is devoted to the study of the activation mechanism of G-protein coupled receptors (GPCRs), one of the largest and most diverse protein families in mammalian genomes, by means of different computer-based simulation techniques. Using force field based classical molecular dynamics (MD), the time evolution of two prototypical GPCRs, rhodopsin and β adrenergic receptors, has been followed for microseconds under different external conditions. Within this approach, the non-native modifications induced by the different engineering techniques used to crystallize most of the known GPCR structures have been identified and some of the mechanisms through which these receptors have managed to optimize their function through evolution have been described and quantified. In detail, this has led to suggest a possible binding mode of agonists to β adrenergic receptors and to identify crucial micro-switches during receptor activation, as well as to describe an asymmetric pathway in the rhodopsin dimer that suggests oligomerization in GPCRs as a biological strategy to enhance activation efficiency. The increased knowledge of GPCR activation mechanism obtained from the classical molecular dynamics simulations hinted to a crucial role of chemical reactions inside the binding pocket of the receptors during the activation cycle. These events have been studied in this thesis using a hybrid quantum mechanics/molecular mechanics (QM/MM) protocol based on Density Functional Theory. Using this approach, it has been possible to characterize the optical properties of the different intermediates along the activation pathway of rhodopsin and to quantitatively estimate the barrier for the proton transfer reaction that induces active state formation. Finally, a novel activation mechanism in diffusible ligands class A GPCRs that indicates proton transfer from the bound ligand to the receptor as a crucial step to reach the active conformation is proposed. Keywords: G-Protein Coupled Receptors (GPCR), Rhodopsin, β Adrenergic Receptor, Molecular Dynamics (MD), Density Functional Theory (DFT).

Abstract Questa tesi é dedicata allo studio dei meccanismi di attivazione dei recettori accoppiati alle proteine G (GPCR), una delle più numerose e variegate famiglie di proteine nell’intero genoma dei mammiferi, tramite l’utilizzo di differenti metodi computazionali. Attraverso l’uso di dinamica molecolare classica con potenziali parametrizzati, é stato possibile studiare l’evoluzione temporale di due prototipici GPCR, rodopsina e recettori β adrenergici, per microsecondi in differenti condizioni esterne. Con questo approccio, é stato possibile identificare le alterazioni introdotte dalle varie tecniche di ingegnerizzazione usate per cristallizzare la maggior parte dei GPCR, nonchè descrivere e quantificare alcuni dei processi attraverso cui questi recettori sono riusciti a ottimizare il loro funzionamento. In dettaglio, ciò ha permesso di descrivere i particolari dell’interazione tra agonisti e recettori β adrenergici e di identificare una serie di “micro-interruttori” durante l’attivazione di questi recettori, e di descrivere un meccanismo di attivazione asimmetrico nel dimero della rodopsina che suggerisce come l’oligomerizzazione nei GPCR possa essere una strategia biologica per aumentarne l’efficienza. Tali informazioni hanno suggerito come alcune reazioni chimiche all’interno del sito attivo dei GPCR potessero svolgere un ruolo cruciale nel processo di attivazione. Questi eventi sono stati studiati utilizzando un protocollo in grado di combinare meccanica classica e quantistica, trattando la parte quantistica con la teoria del funzionale della densità. Con questa tecnica, é stato possibile caratterizare le proprietà ottiche dei differenti stadi durante la fotoattivazione della rodopsina e di quantificare la barriera in energia libera della reazione di trasferimento di protone che anticipa la formazione dello stato attivato. Infine, un trasferimento di protone dall’agonista al recettore é stato proposto come un ingrediente cruciale nel meccanismo di attivazione nei GPCR. Parole chiave: Recettori accoppiati alle Proteine G (GPCR), Rodopsina, Recettori β Adrenergici, Dinamica Molecolare (MD), Teoria del Funzionale della Densità (DFT).

Contents List of Figures

ix

List of Tables

xiii

Glossary

xv

1 Introduction

1

2 G-protein coupled receptors

5

2.1

Rhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.2

β adrenergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3 Theory 3.1

17

Many-body Hamiltonian . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.1.1

Born-Oppenheimer approximation . . . . . . . . . . . . . . . . . . . . . . 18

3.1.2

Car-Parrinello Lagrangian . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.1.3

Empirical force fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.1.4

Density functional theory . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

3.1.5

3.1.4.1

The Hohenberg-Kohn theorems . . . . . . . . . . . . . . . . . . . 22

3.1.4.2

The Kohn-Sham equations . . . . . . . . . . . . . . . . . . . . . 23

3.1.4.3

Exchange-correlation functionals . . . . . . . . . . . . . . . . . . 24

QM/MM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.1.5.1

3.2

Interface Hamiltonian . . . . . . . . . . . . . . . . . . . . . . . . 26

Molecular dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.2.1

Integration of the equations of motion . . . . . . . . . . . . . . . . . . . . 29

3.2.2

Periodic boundary conditions . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.2.3

Thermostats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3.2.4

Barostats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

v

CONTENTS

3.2.5

Calculation of free-energy profiles . . . . . . . . . . . . . . . . . . . . . . . 34

4 Observation of ionic lock formation in molecular dynamics simulations of wild type β1 and β2 adrenergic receptors 37 4.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.2

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.3

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

4.4

4.3.1

Native structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

4.3.2

Binding mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4.3.3

Receptor specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

4.3.4

Activation/inhibition model . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

5 A conserved protonation-induced switch can trigger ionic lock formation in adrenergic receptors 57 5.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

5.2

Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

5.3

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

5.4

5.3.1

Asp(2.50) acidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

5.3.2

Ionic lock configuration is correlated with protonation state of Asp(2.50) . 65

5.3.3

Signal transduction from Asp(2.50) to the cytoplasmatic side . . . . . . . 71

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

6 Predicting novel binding modes of agonists to β adrenergic receptors using all-atom molecular dynamics simulations 77 6.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

6.2

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

6.3

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 6.3.1

6.3.2 6.4

Orthosteric binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 6.3.1.1

Agonist interactions with helices V-VI . . . . . . . . . . . . . . . 84

6.3.1.2

Agonist interactions with helices II, III and VII . . . . . . . . . 86

Extracellular loops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

7 Computational evidence of agonist-induced proton transfer in the binding pocket of β adrenergic receptors 95

vi

CONTENTS

8 Role of aggregation in rhodopsin signal transduction

105

8.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

8.2

Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

8.3

8.4

8.2.1

Molecular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

8.2.2

MD simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 8.3.1

Step 1: Early events in the signal transduction pathway . . . . . . . . . . 111

8.3.2

Step 2: Signal transduction through the protein-protein interface . . . . . 113

8.3.3

Step 3: Asymmetric activation of rhodopsin dimer . . . . . . . . . . . . . 116

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

9 Structural and optical characterization of rhodopsin early photointermediates 123 9.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

9.2

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

9.3

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

9.4

9.3.1

Structural characterization of rhodopsin photointermediates . . . . . . . . 127

9.3.2

Optical characterization of rhodopsin photointermediates . . . . . . . . . 129

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

10 First-principles QM/MM investigation of Schiff base deprotonation in the late rhodopsin photointermediates 133 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 10.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 10.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 10.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 11 Conclusions and outlook

143

References

147

Publications and Presentations

157

Curriculum Vitae

161

vii