Structural Connection Between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling
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Article Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling Graphical Abstract Authors Kate L. White, Matthew T. Eddy, Zhan-Guo Gao, ..., A AR A AR-D2.50N Kenneth A. Jacobson, 2A 2A Vsevolod Katritch, Raymond C. Stevens Na+ Na+ Na+ Na+ Correspondence Na+ Na+ Na+ Na+ [email protected] II II In Brief White and Eddy et al. report agonist- V Na+ I V I bound structures of human A2AAR variants that disrupt allosteric sodium VI VII VII effects. The structures reveal changes in VI hydrogen bonding near a conserved III III activation motif that correspond to VIII VIII striking differences in signaling, providing Na+ Na+ G-protein Na+ Na+ a rationale for increased variant receptor Na+ Na+ stability. Highlights 2.50 3.39 d X-ray structures of A2AAR variants D N and S A agonist complexes 2.50 d A2AAR-D N shows striking loss of G-protein signaling d Structural changes near activation motif correspond to loss of signaling d D2.50N improves GPCR stability for accelerating drug discovery White et al., 2018, Structure 26, 1–11 February 6, 2018 ª 2018 Elsevier Ltd. https://doi.org/10.1016/j.str.2017.12.013 Please cite this article in press as: White et al., Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling, Structure (2018), https://doi.org/10.1016/j.str.2017.12.013 Structure Article Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling Kate L. White,1,3 Matthew T. Eddy,1,3 Zhan-Guo Gao,2 Gye Won Han,1 Tiffany Lian,1 Alexander Deary,1 Nilkanth Patel,1 Kenneth A. Jacobson,2 Vsevolod Katritch,1 and Raymond C. Stevens1,4,* 1Departments of Biological Sciences and Chemistry, Bridge Institute, USC Michelson Center, University of Southern California, 1002 West Childs Way, Los Angeles, CA 90089, USA 2Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA 3These authors contributed equally 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.str.2017.12.013 SUMMARY Tera´ n et al., 2013), dopamine receptors (Neve, 1991), and others as reviewed in the literature (Katritch et al., 2014). Sodium ions are endogenous allosteric modulators Allosteric effects of sodium have been found to be largely of many G-protein-coupled receptors (GPCRs). Mu- mediated by the negatively charged residue D2.50 (Ceresa and tation of key residues in the sodium binding motif Limbird, 1994; Fenalti et al., 2014; Katritch et al., 2014; Liu causes a striking effect on G-protein signaling. We et al., 2012; Massink et al., 2015; Werling et al., 1986) located report the crystal structures of agonist complexes in transmembrane helix II (superscripts indicate the Balles- for two variants in the first sodium coordination teros-Weinstein nomenclature; Ballesteros and Weinstein, 1995). D2.50 is one of the most highly conserved amino acids in shell of the human A2A adenosine receptor, 2.50 2.50 3.39 class A GPCRs (Katritch et al., 2014). Replacement of D D52 N and S91 A. Both structures present an with a neutral amino acid diminished the ability of sodium to overall active-like conformation; however, the vari- compete with agonist binding and concurrently affected G-pro- ants show key changes in the activation motif tein signaling for more than 25 different GPCRs (Hishinuma et al., NPxxY. Changes in the hydrogen bonding network 2017; Katritch et al., 2014). In the d-opioid receptor (DOR), a D2.50 in this microswitch suggest a possible mechanism variant switched the efficacy of an antagonist to an arrestin- for modified G-protein signaling and enhanced ther- biased agonist (Fenalti et al., 2014). For the human A2A adeno- 2.50 mal stability. These structures, signaling data, and sine receptor (A2AAR), replacement of D with alanine thermal stability analysis with a panel of pharmaco- completely abolished G-protein-dependent signaling (Massink et al., 2015). logical ligands provide a basis for understanding 2.50 the role of the sodium-coordinating residues on sta- D forms the core of a cluster of residues that coordinate a sodium ion that was first observed in a high-resolution crystal bility and G-protein signaling. Utilizing the D2.50N structure of A2AAR in complex with an antagonist (Liu et al., variant is a promising method for stabilizing class 2012) and subsequently observed in a DOR-antagonist complex A GPCRs to accelerate structural efforts and drug (Fenalti et al., 2014). In those structures, other residues coordi- discovery. nating sodium are S3.39, which also directly coordinates sodium, and W6.48,N7.49,N7.45, and S7.46, which indirectly coordinate so- dium through a network of water molecules (Figure 1). All of these INTRODUCTION sodium-coordinating residues are highly conserved among class A GPCRs (Katritch et al., 2014). In A2AAR, replacement of any of Endogenous allosteric modulators of G-protein-coupled recep- these sodium pocket residues with alanine altered agonist- tors (GPCRs) are important effectors of receptor signaling and induced G-protein signaling (Massink et al., 2015). include lipids, ions, and intracellular proteins (Changeux and Sodium-coordinating residues also strongly influence thermal Christopoulos, 2016; van der Westhuizen et al., 2015). In partic- unfolding temperatures of GPCRs. Replacement of one or more ular, sodium ions have been proposed to be an endogenous of the sodium-coordinating residues increased denaturation tem- allosteric modulator for many GPCRs (Katritch et al., 2014). Ob- peratures for several GPCRs, including the neurotensin receptor servations in mouse brain extracts over 40 years ago first (NTSR1) (Shibata et al., 2009), k-opioid receptor (KOR) (Schutz demonstrated the allosteric effects of sodium on opioid recep- et al., 2016), and a1a adrenergic receptor (Dodevski and Pluck- tors (Pert et al., 1973). Since those initial observations, sodium’s thun, 2011). For the purinergic P2Y1 receptor (P2Y1R), replace- allosteric effects have been reported from in vitro and in vivo ment of an endogenous D7.49 with asparagine improved assays for over 15 GPCRs, including adrenergic receptors (Cer- protein expression yield and was required for crystallization of a esa and Limbird, 1994), adenosine receptors (Gutie´ rrez-de- P2Y1R-antagonist complexes (Zhang et al., 2015a). Recently, Structure 26, 1–11, February 6, 2018 ª 2018 Elsevier Ltd. 1 Please cite this article in press as: White et al., Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling, Structure (2018), https://doi.org/10.1016/j.str.2017.12.013 AB to directly coordinate the sodium ion (Figure 1)(Liu et al., 2012). Thus, we focused on variants of these two residues in the current study. We measured ligand binding with A2AAR and the variants A2AARÀD52N and A2AARÀS91A with the antagonist ZM241385 and agonist NECA (Table 1). A2AAR, A2AARÀD52N, and A2AARÀS91A all showed similar affinity for the antagonist ZM241385 and agonists CGS21680 and UK432097 (Table 1 and Figure 2A). Affinity of the agonist NECA was slightly higher for the A2AARÀD52N variant (KD 9.16 nM, logKD À8.03 ± 0.10) relative to A2AAR (KD 23.4 nM, logKD À7.63 ± 0.11) and A2AARÀS91A (KD 132 nM, logKD À6.88 ± 0.08). To investigate the allosteric role of sodium on agonist binding, we performed the same binding assays in the presence of a physiologically relevant sodium concentration (150 mM). The presence of so- Figure 1. Allosteric Sodium Binding Pocket and Sodium-Coordi- dium decreased the affinity of the agonist NECA for A AR and ˚ 2A nating Residues in the 1.8 A A2AAR Crystal Structure had no effect on agonist affinity for A2AARÀD52N and (A) Side view of the overall crystal structure of A2AAR in complex with the A2AARÀS91A (Table 1). antagonist ZM241385. A2AAR is shown as a cartoon representation and ZM241385 is shown as a blue stick representation (PDB: 4EIY) (Liu et al., We examined G-protein signaling for the A2AAR variants by 2012). Water molecules are shown as red spheres and sodium is shown as a measuring cAMP accumulation via receptor activation upon blue sphere. stimulation with the agonist UK432097. A2AARÀD52N evi- (B) Expansion of the allosteric sodium pocket. Sodium is shown near the denced no cAMP accumulation upon UK432097 stimulation, center as a blue sphere and water molecules are shown as red spheres. similar to what was observed with A2AARÀD52A in an earlier Residues coordinating with the sodium ion or nearby water molecules are À labeled. Dashed lines indicate polar contacts and helices are labeled with report (Massink et al., 2015), while A2AAR S91A had similar À Roman numerals. activity to A2AAR (Figure 2B and Table 1). While A2AAR S91A signaling was previously found to have higher basal and lower Emax signaling upon CGS21680 agonist stimulation (Massink observations in A2AAR by nuclear magnetic resonance (NMR) et al., 2015), differences reported here may be due to using spectroscopy showed that replacement of D2.50 with asparagine different cell types, agonists, and assay conditions (for example, drastically altered conformational dynamics at the A2AAR intracel- adenosine deaminase was included in our experiments to elimi- lular surface without altering the receptor’s conformation at the nate the influence of endogenous adenosine). extracellular surface (Eddy et al., 2017). Motivated by these numerous observations, we sought to pro- Crystal Structures of A2AAR Sodium-Site Variants in vide a structural basis for how the sodium-coordinating residues Complex with an Agonist Present an Overall Active-like regulate G-protein signaling and affect denaturation tempera- Conformation tures of human A2AAR. A2AAR is a validated drug target for Par- We determined the crystal structures of A2AARÀD52N and kinson’s disease (Cieslak et al., 2008) and chronic obstructive A2AARÀS91A in complex with the full agonist UK432097.