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Novel Allosteric Communication in Nuclear Receptor Activation

Kohn, Jeffrey (2016)
Dissertation (182 pages)
Committee Chair / Thesis Adviser: Ortlund, Eric
Committee Members: Dunham, Christine ; Murphy, T J ; Sutliff, Roy
Research Fields: Biochemistry
Keywords: Nuclear receptors; Gene regulation; Phospholipids; Corticosteroids; Allostery
Program: Laney Graduate School, Biological and Biomedical Sciences (Molecular & Systems Pharmacology)
Permanent url: http://pid.emory.edu/ark:/25593/rgzpc

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

Cell culture ............................................................................................................................ 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

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