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Historical Mechanisms Driving the Evolution of Ligand Specificity in Steroid Hormone Nuclear Receptors

Colucci, Jennifer (2014)
Dissertation (242 pages)
Committee Chair / Thesis Adviser: Ortlund, Eric
Committee Members: Husain, Ahsan ; Wilkinson, Keith D ; Dunham, Christine ; Hepler, John R
Research Fields: Chemistry, Biochemistry; Biology, Molecular; Biology, General
Keywords: X-Ray Crystallography; Evolution; Steroid Receptor
Program: Laney Graduate School, Biological and Biomedical Sciences (Biochemistry, Cell & Developmental Biology)
Permanent url: http://pid.emory.edu/ark:/25593/fzcw3

Abstract

The genetic and biophysical mechanisms by which new protein functions evolve are central concerns in evolutionary biology and molecular evolution. Despite much speculation, we know little about how protein function evolves in natural proteins. Here, we use ancestral protein reconstruction (APR) to trace the evolutionary history of ligand recognition in steroid hormone nuclear receptors (SRs), an ancient family of ligand-regulated transcription factors that enable long-range cellular communication central to multicellular life. We found that the most ancestral SR, ancSR1, was regulated by estrogens (steroids with aromatic A rings and small substituents at their carbon 17 position). After a gene duplication event, the duplicate SR, ancSR2, evolved specificity towards progestagens and corticosteroids (nonaromatic 3-ketosteroids with bulky substituents at their carbon 17 position) while excluding estrogens from the binding pocket. We show that this switch from ancSR1- to ancSR2-specificity is mediated by the evolution of several large-effect substitutions within the ligand binding pocket (LBP) that confer a stable hydrogen-bond network for the A ring of nonaromatic 3-ketosteroids. We show that recognition of the hormone's carbon 17 substituent in ancSR2 was conferred via a series of epistatic interactions that served to reposition the ligand and exploit available hydrogen bond capabilities within the ligand binding pocket. Finally, we show that ancestral receptors can be modulated by modern pharmaceuticals and suggest that the SR antagonist mifepristone may act as receptor modulator at the proteins coactivator binding cleft.

Table of Contents

Distribution Agreement.. 1

ABBREVIATIONS. 13

ABSTRACT.. 16

By Jennifer Katherine Colucci. 16

CHAPTER 1: INTRODUCTION.. 18

Steroid Receptors regulate normal and disease physiologies. 20

Nuclear Receptor structure. 23

SR mechanism of activation. 24

Molecular Evolution. 26

Ancestral Gene Resurrection. 27

Figure 1.3: Nuclear Receptor Domain Architecture. 33

Figure 1.6: Model of the mechanism of NR activation. 36

A. Percent identity of SR LBDs: 38

References. 39

CHAPTER 2: EVOLUTION OF MINIMAL SPECIFICITY AND PROMISCUITY IN STEROID HORMONE RECEPTORS. 47

Abstract. 49

Author Summary. 50

Introduction. 51

Results and Discussion. 54

Reconstruction and characterization of ancestral proteins. 54

Ancestral structure-activity criteria. 56

Minimal specificity in SR evolution. 57

An evolutionary explanation for SR-mediated endocrine disruption. 59

Structural causes of SR promiscuity. 60

Promiscuity, selection, and neutrality in the evolution of signaling. 61

Methods. 63

Phylogenetics and ancestral sequence reconstruction. 63

Reporter activation assays. 64

Alternative ancestral reconstructions. 65

Protein expression. 66

Crystallization and structural analysis. 67

Acknowledgements. 69

Figures. 70

Figure 2.1: Evolutionary expansion of the steroid receptors and their ligands. 70

Figure 2.2: Ligand-recognition rules of ancSR1 and ancSR2. 72

Figure 2.3: Evolution of minimal specificity. 74

Figure 2.4: Structural causes of minimal specificity. 76

Figure 2.5: Histogram of posterior probabilities for ancSR2. 78

Figure 2.6: Histogram of posterior probabilities for ancSR1. 79

Figure 2.7: Dose activation curves of ancSR1. 80

Figure 2.8: Dose activation curves of ancSR2. 81

Figure 2.9: The specificity of ancSR1 is robust to uncertainty in the reconstruction. 82

Figure 2.10 The specificity of ancSR2 is robust to uncertainty in the reconstruction. 83

Figure 2.11: Sensitivities of extant human receptors to an estrogen, androgen, progestagen, and corticosteroid. 84

Figure 2.12: Activation of the estrogen receptor ligand binding domains of two annelids and human ERα. 85

Figure 2.13: AncSR2 is not activated by the nonsteroidal ER agonists diethylstilbestrol and genistein and is not inhibited by ICI182870 and 4-hydroxytamoxifen. 86

Figure 2.14: ML steroid receptor phylogeny for ancSR2. 87

Figure 2.15: ML steroid receptor phylogeny for ancSR1. 88

Figure 2.16: Unreduced 184-taxon steroid receptor gene duplication phylogeny. 90

Figure 2.17: Omit maps of progesterone and 11-deoxycorticosterone. 91

Table 2.1: Reconstructed sequence of ancSR2. 92

Table 2.2: Reconstructed sequence of ancSR1. 93

Table 2.3: ancSR1 and ancSR2 percent similarities. 94

Table 2.4: CID numbers for synthetic and natural steroids used in this study. 95

Table 2.5: Fold preferences for hormone pairs. 96

Table 2.6: Data collection and refinement statistics. 97

Table 2.7: Receptors and organisms used for phylogenetic analyses. 98

Table 2.8: ancSR2 sequence comparison. 99

References. 100

CHAPTER 3: BIOPHYSICAL MECHANISMS FOR LARGE-EFFECT MUTATIONS IN THE EVOLUTION OF STEROID HORMONE RECEPTORS. 105

Introduction. 108

Protein biophysics and evolution. 108

An evolutionary shift in hormone specificity. 109

Results and Discussion. 111

Phylogenetic and structural analyses to identify causal mutations. 111

Two large-effect replacements shifted hormone specificity. 111

Structural mechanisms for the shift in specificity. 112

Changes in the energetic landscape of ligand binding. 113

Experimental analysis of changes in dynamics. 115

Arg82 is necessary for ligand-specificity. 116

Evolution of proteins as complex physical systems. 117

Methods. 119

Reporter activation assays. 119

Sequence conservation analysis. 120

Molecular dynamics methods. 121

Free energy landscapes. 122

Characterization of water-penetrated states. 124

HDX-MS. 124

Acknowledgements. 128

Figures. 129

Figure 3.1: Evolution of ancSR1 and ancSR2 specificity. 129

Figure 3.2: Large-effect historical mutations drove the evolution of new ligand specificity. 132

Figure 3.3: Two historical mutations altered the energetic landscape of protein-ligand binding. 133

Figure 3.4: Ligand-specific disruption of the A-ring hydrogen-bond network. 134

Figure 3.5: Cognate steroids of the six human steroid receptors. 136

Figure 3.6: A-ring ligand contacts are largely conserved between ancSR2 (magenta) and ancSR2 (blue). 137

Figure 3.7: Representative dose activation curves of ancSR2/Q41e/M75l and ancSR2 wild-type. 138

Figure 3.8: Representative dose activation curves of ancSR1 and ancSR1/e41Q/l75M. 139

Figure 3.9: A control MD simulation with the apo protein. 140

Figure 3.10: Derived amino acids introduce a new direct contact with the norP 3-keto group. 141

Figure 3.11 Populated rotamers of Glu41 and Gln41. 142

Figure 3.12: Dependence of number of states on ΔGbarrier. 143

Figure 3.13: Non-aromatized steroid with 3-hydroxyl does not populate frustrated hydrogen bond networks. 144

Figure 3.14: Historical mutations cause increased peptide solvent exchange in a ligand-dependent manner. 145

Figure 3.15: Model fits to incorporation vs. time data for the five peptides which exhibited decreased NPT-specific protection factors in the derived state. 147

Table 3.1: Pubmed compound identifier (CID) numbers for cholesterol and the synthetic and natural steroid hormones tested in this study. 148

Table 3.2: Conservation analysis of extant naSRs and ERs. 154

Table 3.3: Simulations display additivity: absolute free energies of barriers are the same for ij versus j→i transitions. 155

Table 3.4: Transition matrices for top 95% of observed states with 2 kcal/mol energy cutoff. 156

Table 3.5: HDX-MS kinetics model selection. 157

References. 158

CHAPTER 4: X-RAY CRYSTAL STRUCTURE OF THE ANCESTRAL 3-KETOSTEROID RECEPTOR - PROGESTERONE - MIFEPRISTONE COMPLEX SHOWS MIFEPRISTONE BOUND AT THE COACTIVATOR BINDING SURFACE.. 162

Abstract. 163

Introduction. 164

Materials and Methods. 166

Reagents. 166

Expression and Purification. 166

Crystallization, data collection, structure determination and refinement. 167

Reporter Gene Assays. 167

Results. 169

Overall Structure. 169

Mifepristone binds at two distinct surface sites. 170

Improved resolution of the ancSR2-progesterone structure permits visualization of D-ring contacts. 171

Discussion. 172

Acknowledgements. 176

Figures. 177

Figure 4.1: Crystals of the ancSR2-progesterone-mifepristone complex and in vitro activation data. 177

Figure 4.2: Overall structure of the ancSR2-progesterone-mifepristone complex. 178

Figure 4.3: Omit maps of bound ligands. 179

Figure 4.4: Mifepristone binding site interactions. 180

Figure 4.5: Mifepristone occupies the coactivator protein space. 181

Figure 4.6: Global alignment of progesterone-bound steroid receptors. 183

Figure 4.7: Mifepristone and 4-hydroxytamoxifen show similar binding modes to the steroid receptor coactivator binding cleft. 184

References. 185

CHAPTER 5: EXPRESSION, PURIFICATION, AND CRYSTALLIZATION OF THE ANCESTRAL ANDROGEN RECEPTOR-DHT COMPLEX.. 189

Abstract. 190

Introduction. 191

Materials and Methods. 193

Reagents. 193

Cloning. 193

Expression and Purification. 194

Crystallization and Data Collection. 194

Results and Discussion. 196

Acknowledgements. 197

Figures. 198

Figure 5.1: Following a series of affinity columns, ancAR1-DHT was purified to homogeneity. 198

Figure 5.2: Crystals of ancAR1-DHT. 199

Figure 5.3: Diffraction image of an ancAR1-DHT crystal. 200

Table 5.1: Data collection statistics for AncAR1-DHT-Tif2. 201

References. 202

CHAPTER 6: BEYOND MINIMAL SPECIFICITY: EVOLVING THE ABILITY TO DISCRIMINATE AMONG DIVERSE 3-KETOSTEROIDS. 211

Abstract. 212

Introduction. 213

Materials and Methods. 215

Reagents. 215

Structural Analysis. 216

Mutagenesis. 216

Reporter activation assays. 216

Results. 218

Comparison of estrogen versus progesterone recognition in the ligand binding pocket 218

Which amino acid substitutions facilitate recognition of bulky carbon 17 substituents?. 219

Discussion. 221

Figure 6.1: Phylogeny of the Steroid Receptor lineage. 223

Figure 6.2: Rotation of the 17-acetyl ligand in the binding pocket allows for exploitation of pre-existing hydrogen bond capacity. 224

Figure 6.3: Forward Evolution of D-ring residues increased preference for 17-acetyl ligands. 225

Figure 6.4: Epistatic interactions shaped ancSR2 evolution. 226

Figure 6.5: Evolutionary pathway to the evolution of 17-acetyl recognition. 227

Table 6.1: Hormone sensitivity of WT ancSR2 and mutants. 228

References. 229

CHAPTER 7: DISCUSSION.. 231

How did the differences in ligand specificity between the ERs and naSRs evolve? 232

What are the mechanisms that dictate the ligand preferences of ERs and naSRs? 233

How can ancestral proteins be used to understand modern pharmacology? 235

How do epistatic interactions influence the evolution of ligand specificity? 236

Composite discussion. 238

Future Directions. 240

Can we completely recapitulate the switch in hormone selectivity from ancSR1 to ancSR2? 240

What factors contribute to the evolution of androgen specificity? 241

References. 242

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