Novel Allosteric Communication in Nuclear Receptor Activation Pubblico

Abstract

Nuclear receptors (NRs) are highly important targets for the pharmacological management of many human conditions, including cancer, inflammation, autoimmunity, metabolic syndrome, thyroid dysfunction, and reproduction. Unfortunately, NR-targeting drugs often cause significant adverse reactions. NRs are a highly interrelated family of transcription factors that are evolutionarily descended from a common ancestor, from which they inherited a conserved structural fold and mode of activity. NR activation involves the recruitment of coactivator and corepressor proteins, which promote the transcription or repression of the target gene. Thus, side-effects arise from the off-target action of a drug on close relatives of its target, and the inability of the drug to selectively control the transactivation or transrepression of the therapeutically relevant genes. To address these problems, the present work considers the structural biology of the NR family from the perspective of their molecular evolution in order to elucidate the structural mechanisms that drive ligand-regulated receptor activity in two receptors, the corticosteroid receptors (consisting of the glucocorticoid and mineralocorticoid receptors, GR and MR) and liver receptor homolog-1 (LRH-1). In all of these receptors, ligand binding affected, and was affected, by interaction of residues in a flexible region at the bottom of the receptor. In the corticosteroid receptors, mutations at this region toggled the activity of synthetic glucocorticoids between agonist and antagonist, without affecting the activity of endogenous hormones. In LRH-1, which is modulated by phospholipids (PLs), the length of the PL tails differentially stabilized this region, allowing for the selective recruitment of coactivators or corepressors. Molecular dynamics simulations demonstrated that this region was in allosteric communication with the coregulator binding surface, permitting the binding of varying ligands to promote the selective recruitment of coactivators or corepressors. Thus, this region constitutes a novel, alternate activation function in the NR ligand binding domain that may be exploitable by next generation drugs in order to improve their therapeutic profile.

Table of Contents

Chapter 1: Introduction 1

Regulation of large gene programs by nuclear receptors 2

Nuclear receptor structure and function 2

Structure and function of the DNA-binding domain 8

Structure and function of the ligand-binding domain 10

The N-terminal domain and hinge 14

Nuclear receptor coregulators 16

Conservation of nuclear receptor sequence and structure 19

Evolution of protein families and the resurrection of ancient genes 21

Molecular Evolution 21

Ancestral Gene Resurrection 23

NRs studied in this work 26

The corticosteroid receptors 26

Liver receptor homolog-1 30

Current state of nuclear receptor pharmacology 30

Nuclear receptors as pharmaceutical targets 30

Questions and hypotheses addressed in this work 31

References 33

Chapter 2: Phospholipid-driven gene regulation 42

Summary 42

Introduction 43

Phospholipids 43

PLs outside the membrane 45

PLs are a new class of hormone 45

Nuclear Receptors: lipid regulated transcription factors 45

Nuclear receptor structure and function 45

PL-driven NR activation 46

Case Studies 47

LRH-1 47

Bound E. coli PLs offer the first clue that LRH-1 may be PL regulated 48

LRH-1 - PIP interactions 49

DLPC 49

SF-1 50

E. coli PL binding from early structural studies 51

PA versus sphingosine 51

PIP2 versus PIP3 51

PPARs 52

PPARα and PC 16:0/18:1 52

PPARγ and tail-oxidized PLs 53

USP 54

E. Coli PLs 54

PL transport and PL dependent coactivation 54

PPAR and PC-TP 54

Structural Analysis of PL binding proteins 55

What does it take to bind to PLs as a ligand? 55

Shuttlers versus transcription factors 55

Parallels in the immune system 57

Comparison to the PL PI/PC transporter Sec14 57

PL presentation as a model for PL dependent signaling 58

Closing Remarks 58

References 61

Chapter 3: Unexpected allosteric network contributes to LRH-1 co-regulator selectivity 67

Summary 67

Introduction 68

Experimental Procedures 70

Reagents 70

Protein expression and purification 70

Structure determination 70

Local Conformational Analysis 71

Synthesis of NBD-DLPE 72

Phospholipid binding assays 72

Reporter gene assays 73

Model Construction for Molecular Dynamics 73

Molecular Dynamics 74

Analysis methodology 75

Results 76

Structure of the apo LRH-1 LBD - TIF complex: 76

Improved structure of the LRH-1 LBD - E. coli PL - TIF2 complex 76

Co-regulator binding interactions are altered by ligand status 84

Ligand and coregulator drive differential effects on local residue environment 87

The Activated LRH-1 Complex Exhibits Coordinated Motions 89

MD Simulations Demonstrate Communication between β-sheet-H6 and the AF-H through Helices 3, 4, and 5 91

Structural and Dynamical Rationale for Lipid and Co-regulator Agreement 92

Modest disruption of interhelical interactions along the allosteric pathway reduces, but does not eliminate, LRH-1 activity 98

Discussion 101

Lipid mediated allosteric control of a protein-protein binding interface 102

References 104

Chapter 4: Regulation of LRH-1 by endogenous lipids - preliminary findings 109

Introduction 110

Experimental Procedures 111

Phospholipid pulldown 111

LRH-1 Phospholipid binding assay 111

LRH-1 activation assay 111

Expression of PCTP 112

Loading of PCTP with PC-NBD probe 112

PCTP--LRH-1 direct phospholipid transfer assay 113

Results 113

Discussion 118

References 123

Chapter 5: Deciphering modern glucocorticoid cross-pharmacology using ancestral corticosteroid receptors 125

Summary 125

Introduction 126

Experimental Procedures 128

Protein expression and purification 128

Crystallization, data collection, structural refinement 128

Mutagenesis 129

Ligand binding assays 129

In-cell activation assays 129

Results 130

AncGR2-TIF2-MOF crystal structure 130

Structural and evolutionary basis for PR cross-reactivity 133

Structural and evolutionary basis for selectivity against MR 133

A single residue controls MOF selectivity and transcriptional activity 138

Discussion 143

References 144

Chapter 6: Discussion 147

Conclusions 148

Phospholipids are a novel class of ligands 148

PL modular structure permits fine control of coregulator recruitment 148

Evolution of protein structure explains drug selectivity 149

Identification of a novel activation function in allosteric communication with the AF-2 surface 152

Remaining questions and future directions 156

LRH-1 remains an untapped pharmaceutical target 156

Evolutionary context enhances our understanding of biological systems 157

Closing remarks: the future of nuclear receptor pharmacology 158

References 160

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Parola chiave	Gene regulation Nuclear receptors Corticosteroids Allostery Phospholipids
Committee Chair / Thesis Advisor	Ortlund, Eric, Emory University
Committee Members	Sutliff, Roy, Emory University Murphy, T J, Emory University Dunham, Christine, Emory University

Ultima modifica

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Novel Allosteric Communication in Nuclear Receptor Activation ()	2018-08-28 15:25:56 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions