EPR study of ligand-receptor interactions

Abstract

Ligand-receptor interactions are powerful determinants of biochemical pathways. In this work, the role of ligand binding and coregulator interaction is examined in particular as it related to changes in receptor dynamics and conformation. The emerging technique of site directed spin labeling and electron paramagnetic resonance (EPR) is applied to investigate ligand-induced effects on estrogen receptor (ER), a pharmaceutically relevant member of the nuclear receptor superfamily of transcription factors. Chapter 1 introduces the relevant background information on the biology, structure and physiology of NRs, with emphasis on the alpha isoform of the receptor (ER-α,). An overview of the current state of the field is presented. This chapter also introduces application of EPR in the context of biological investigation of proteins dynamics and structure The specific techniques and methods used in this study are explored in detail in chapter 2. Here the rationale of combining estradiol ligand substituted at the 11β, position with spin labeling of helix 12 of ER-α, is discussed. This chapter provides experimental details regarding ER mutant production and characterization. Relevant details about EPR theory and lineshape analysis are explored. In this chapter, double electron-electron resonance (DEER) is discussed by introducing both a theoretical and experimental topics. In chapter 3, the dynamic response of the human ER-α, ligand binding domain (ERα,-LBD, residues 302-552) to the binding of different ligands and coactivator peptides is investigated by site-directed spin labeling EPR (SDSL-EPR). Specific labeling at residue 543 of the C-terminal helix 12 (H12) domain has provided the first direct experimental demonstration that this domain undergoes dynamic changes in response to ligand binding that correlate with the ligand's biological activity. Ligand-dependent changes are also observable for a label positioned at residue 530 in the H12 hinge region, however much more dramatic changes are observed at this position in the presence of both ligand and coregulator peptides. We investigated the ligand/coregulator induced changes in structure by measuring interspin distances in 530 labeled ERα,-LBD dimers using DEER. These results extend the current model of ERα,-LBD action and provide dynamic information on the H12 region as well as quantitative structural information on the dimer conformation of ERα,-LBD in solution. The results suggest a large-scale remodeling of the ER dimer complex that is sensitive to details of the ligand structure as well as the nature of the coactivator sequence. In chapter 4 two nitroxide labeled estradiols, HO-2105 and HO-2447 are characterized as new molecular probes of ligand/receptor interaction with the ERα,-LBD. EPR spectroscopy was used to investigate the binding properties and local dynamics of the spin-labeled ligand. Fluorescence spectroscopy demonstrated quenching of both the fluorescence of estradiol and of the intrinsic tryptophan fluorescence of ERα,-LBD by the nitroxide moiety of the labeled ligand. We describe two methods to assay binding of the probes to ERα,-LBD: (i) an EPR derived binding assay and (ii) an intrinsic tryptophan fluorescence quenching binding assay. Saturation binding studies of the estrogen probes using the two assays showed good agreement between the independent techniques. DEER spectroscopy was used to measure interspin distances between the bound probes in the ERα,-LBD homodimer complex. The structural results are consistent with X-ray crystallography of the ERα,-LBD dimer and provide new information about the distribution of conformations in the homodimer. The spin labeled estradiols described here serve as versatile probes of ligand binding, local dynamics and structure with potential applications as ER selective imaging agents and as oxidative stress probes. Chapter 5 includes results obtained on other projects not related to ER, but all sharing the broader theme of application of EPR to study biophysical systems. EPR is a versatile technique that enables study of a variety of biophysical phenomena. Here we describe applications of EPR as a method to evaluate singlet oxygen (1O2), production, characterize conformational effects of hydrophobic mismatch on transmembrane helices, develop methods for characterizing protein self-assembly and characterize unstructured protein domains. Furthermore, as these projects result from collaborations with research groups from different disciplines such as organic chemistry, medicinal chemistry, biochemistry and mechanical engineering they each add a particular set of challenges. Chapter 6 evaluates future directions for continuing the investigation into the molecular basis of ER action. Here we develop a theoretical model for the effect of ligand/coregulator interaction on ER that is based on the combined results previously presented. We also consider foreseeable challenges and provide recommendations relative to experimental design on new spin labeled ER investigations.