1.3 The strategy for cell-free synthesis of GPCRs applied in this study
1.3.4 The GPCRs analysed in this study
1-adrenergic receptors
The -adrenergic receptors ( AR) are key players in the mediation of sympathetic nervous system regulated cardiovascular functions. Three receptor subtypes called ( 1 - 3AR) were identified in human and pharmacologically characterised (Post et al. 1999). Cardiovascular functions are mainly regulated through 1- and 2AR, while 3AR is involved in the metabolism of adipose tissues. Through binding of catecholamine hormones in the cardiovascular tissues, AR activate cAMP coupled downstream signalling events that are responsible for increased heart beating rates (chronotropism), force of cardiac contraction (inotropism), the rate of cardiac relaxation (lusitropism) and automaticity (Post et al. 1999).
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Disturbances in AR signalling pathways are associated with severe diseases like congestive heart failure and a large group of drugs called -blockers are directly addressing ARs in the cardiovascular system (Post et al. 1999, Fisker et al. 2015).
In relation to its high pharmacological importance, ARs are in the main focus of structural analysis and, after photo-rhodopsin, 2- and 1AR were the second and third distinct GPCRs from which high-resolution structures could be obtained. Human 2AR could be crystallised in lipidic cubic phase with an inverse agonist bound after insertion of T4-lysozyme in the ICL 3 (Cherezov et al. 2007). Human 1AR is highly sensitive to detergents and high temperatures (Serrano-Vega and Tate 2009) and crystallisation trials were therefore focused on the more robust turkey 1AR, which could be crystallised in octylthioglucoside with a ligand after N- and C-terminal and ICL 3 truncation and introduction of eight thermostabilising mutations (Warne et al. 2008) (Fig. 1.7).
Fig. 1.7 Structures of a 1-adrenergic receptor and its ligands.
Left: Thermostabilised turkey 1AR bound to cyanopindolol was the third GPCR that could be crystallised in high resolution. Right: The pronounced pharmacological profile of 1AR includes full and partial agonists as well as inverse and biased agonists. Figures adapted from Warne et al. 2008 and Warne and Tate 2013.
1AR has a pronounced pharmacological profile and a set of both full and partial agonists exist as well as inverse and biased agonists (Fig. 1.7). Up to date, 18 distinct structures of turkey 1AR are openly available, with bound inverse agonists, (neutral) antagonists and partial agonists as well as a structure crystallised without any ligand (Isberg et al. 2016). This profound data set helped in identification of critical determinants of ligand efficacy and biased signalling, which are based on differences in the contraction of the ligand binding pocket and a change in rotameric conformation of Ser 5.46 upon formation of a hydrogen bond with the ligand (Warne and Tate 2013).
The extensive structural information as well as high stability through thermostabilising mutations were the main reasons for the CFPS trials performed in the present study. A schematical overview on the constructs is given in Fig. 1.8. Mainly, a turkey 1AR construct was analysed containing deletions in the N- and C-terminus and in the ICL 3, with and without 11 point mutations, making the receptor highly thermostable. The thermostabilised construct was highly similar to a previously published design, where three additional mutations resulted in an increased stability compared to the 1AR construct used for initial crystallisation (Miller and Tate 2011). DNA templates for stabilised and non-stabilised turkey
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1AR were kindly provided by Ali Jazayeri (Heptares Therapeutics, Welwyn Garden City, UK).
Furthermore, human 1AR was analysed in this study with thermostabilising mutations and truncations adapted from the turkey receptor and as full-length construct. Human 1AR has a sequence similarity of 76% with turkey 1AR, excluding the N- and C-terminal regions (Serrano-Vega and Tate 2009).
Fig. 1.8 1-adrenergic receptor constructs analysed in this thesis.
The full-length turkey 1AR depicted on the left side was modified by truncations of the N- and C-terminus and in the ICL 3 and by introduction of 11 point mutations, making the receptor highly thermostable (top right). Human 1AR has a high sequence identity to the turkey variant and was analysed as a full-length thermostabilised truncated thermostabilised and truncated non-stabilised construct in this thesis (bottom right).
Receptors of the Endothelin system
The Endothelin system mediates the strongest vasoconstriction effects found so far in humans and other mammals (Alexander et al. 2015). It is pharmacologically addressed for the treatment of pulmonary arterial hypertension, chronic kidney disease, chronic heart failure and others. In humans, physiological effects are primarily mediated by three 21-amino acid long peptides called Endothelin 1 - 3 (ET-1 - 3), acting on the two Endothelin receptors type A and B (ETA and ETB). While ETA receptor shows and order of magnitude higher specificity to ET-1 and ET-2 than for ET-3, binding of all three Endothelins to ETB receptor is equally potent (Alexander et al. 2015). ET-1 is the most abundant Endothelin isoform in the human cardiovascular system. After its release from the endothelium, ET-1 mediates vasoconstriction mainly in a paracrine manner via ETA receptors in the adjacent smooth muscle cells, where phospholipase C mediated downstream signalling events cause Ca2+ influx and subsequent constriction (Vignon-Zellweger et al. 2012). Autocrine effects of ET-1 on ETB receptor the endothelium cells cause vasodilatation in the vascular system by nitric oxide release.
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Additionally, ETB acts as a clearing receptor that binds, internalizes and removes ET-1 from circulation in the intercellular space (Vignon-Zellweger et al. 2012). Additional tissue distributions and functions have been reported for both receptors, with highest mRNA expression levels of ETB found in brain tissues that might be associated with high ET-3 levels in this body part (Alexander et al. 2015).
High ETB receptor concentrations were also found in the kidney, where the Endothelin system contributes to renal hemodynamics and regulation of glomerular pressure (Vignon-Zellweger et al.
2012). ET-2 shows high pharmacological similarities to ET-1 and might have compensatory effects.
Key roles in ovarian physiology and ovulation as well as chemokine roles in the inflammatory system are further debated (Alexander et al. 2015).
From a molecular and structural perspective, all three Endothelins are 21-amino acid long peptides that share sequence homology in the seven C-terminal amino acids. Two intramolecular disulphide bonds are further common characteristics, while the N-terminal part, which adapts -helical shape in all three Endothelins, shows sequence alterations, in particular for the ET-3 peptide (Fig. 1.9).
Fig. 1.9 Structural details of Endothelins and Endothelin receptors.
Left: Endothelin 1 - 3 show high sequence homology in the C-terminal region and common disulphide bridge formation, while the N-terminal region is altered, in particular in ET-3 (blue). Green stars indicate residues that are thought to form a stripe due secondary helical structure arrangements and red dots indicate potential sites for radiolabelling with 125I. Right: The crystal structures of ETB with and without bound ET-1 offer structural insights in the binding affinity and specificity of the Endothelin system. Figures adapted from Davenport et al. 2016 and Shihoya et al. 2016.
Ligand specificity towards the two Endothelin receptors is mediated by the N-terminal part of the peptide, with Endothelin-3 having an order of magnitude lower affinity to ETA receptor, as mentioned earlier. The disulphide bridges have further effects on the ligand specificity and affinity of all three Endothelins to ETA is drastically reduced when they are abolished, while binding to ETB receptor is unimpaired (Tam et al. 1994, Lättig et al. 2009).
Two recently published crystal structures reveal structural insights in the binding mechanism of ET-1 to ETB receptor (Shihoya et al. 20ET-16). The ligand is forming a question mark structure with the
C-20
terminus deeply inserted in the ligand binding cavity formed by the TMS of the ETB receptor (Fig. 1.9).
An exceptional role for high affinity binding is mediated by the C-terminal Trp in ET-1 and mutations of this amino acid completely abolish ligand binding (Lättig et al. 2009). The curved region in the ET-1 question mark structure is pointing towards and interacting with the ECL 2 and N-term region of ETB and, although no high resolution structure of ETA is available to date, this interaction is thought to mediate ligand specificity, in accordance with the sequence alteration in ET-3 in this region (Lättig et al. 2009, Shihoya et al. 2016).
Not least because of their interesting ligand specificity and high physiological relevance, both ETA and ETB receptor were previously analysed by CFPS techniques (Klammt et al. 2007, Junge et al. 2010, Proverbio et al. 2013). So far, best production conditions of ETA receptor in a E. coli based CFPS system were found by P-CF-synthesis, solubilisation in LPPG and subsequent detergent exchange to either Brij-35 or Fos-16. Receptor samples were found to be homogeneous on SEC and ligand binding competent, although with significantly lower affinity than previously published data (Junge et al. 2010). For ETB receptor, so far best results were obtained by supplementation of ND (DMPC) in the CF-synthesis reaction. A distinct ligand binding behaviour could be observed by SPR-techniques, with affinities in the nanomolar range. However, upon radioligand binding assays, only minor fractions of the receptor were found to be ligand binding competent (Proverbio et al. 2013). Further ETB receptor synthesis in a an insect cell based CFPS system with ER-derived microsomes showed similar low folding efficiencies, which were accounted mainly for shortcomings of the membrane insertion machineries of the microsomes (Merk et al. 2015).
In this thesis, CFPS of both ETA and ETB receptor was studied (Fig. 1.10). The focus was on the ETB receptor, which was synthesised with a N-terminal deletion of the first 26 amino acids, which form a putative signal sequence and are truncated in vivo during receptor maturation and localisation at the plasma membrane (Saito et al. 1991). Furthermore, thermostabilised ETB receptor construct ranging from amino acid position 27 to 401 was analysed, as well as the full-length ETA receptor, which shows 63% sequence similarity with ETB receptor (Davenport et al. 2016).
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Fig. 1.10 Endothelin receptors analysed in this thesis.
ETB receptor was analysed as a construct without the 26 amino acid long N-terminal putative signal sequence and as a thermostabilised truncation construct. ETA receptor was analysed as a full-length construct, which shows 63%
sequence similarity to ETB.
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