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Bias-Inducing Allosteric Binding Site in Mu-Opioid Receptor Signaling

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Bias-Inducing Allosteric Binding Site in Mu-Opioid Receptor Signaling Research Article Bias‑inducing allosteric binding site in mu‑opioid receptor signaling Andrés F. Marmolejo‑Valencia1 · Abraham Madariaga‑Mazón1 · Karina Martinez‑Mayorga1 Received: 23 January 2021 / Accepted: 12 March 2021 © The Author(s) 2021 OPEN Abstract G-protein-biased agonism of the mu-opioid receptor (μ-OR) is emerging as a promising strategy in analgesia. A deep understanding of how biased agonists modulate and diferentiate G-protein-coupled receptors (GPCR) signaling path- ways and how this is transferred into the cell are open questions. Here, using extensive all-atom molecular dynamics simulations, we analyzed the binding recognition process and signaling efects of three prototype μ-OR agonists. Our suggested structural mechanism of biased signaling in μ-OR involves an allosteric sodium ion site, water networks, confor- mational rearrangements in conserved motifs and collective motions of loops and transmembrane helices. These analyses led us to highlight the relevance of a bias-inducing allosteric binding site in the understanding of μ-OR’s G-protein-biased signaling. These results also suggest a competitive equilibrium between the agonists and the allosteric sodium ion, where the bias-inducing allosteric binding site can be modulated by this ion or an agonist such as herkinorin. Notably, herkinorin arises as the archetype modulator of μ-OR and its interactive pattern could be used for screening eforts via protein–ligand interaction fngerprint (PLIF) studies. Article highlights • Agonists and a sodium ion compete for the bias-inducing allosteric binding site that modulates signaling in mu-opioid receptors. • Molecular dynamics simulations of the prototype μ-OR agonist suggest a competitive equilibrium involving the agonist and an allosteric sodium ion. • Analysis of experimental data from the literature and molecular models provides the structural bases of biased ago- nism on μ-OR. Keywords Biased signaling · GPCR · Mu-opioid receptor · Molecular dynamics simulations 1 Introduction G-proteins and/or with β-arrestins. Preferential activation to either pathway is possible, leading to diferent physi- G-protein-coupled receptors (GPCRs) are seven-trans- ological efects. Agonists that have this ability are called membrane (TM) helical proteins that trigger signaling biased agonists, and the efect is named functional selec- pathways, mediated by interactions with heterotrimeric tivity. Other modes of GPCR activation are intracellular Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s42452- 021- 04505-8. * Karina Martinez-Mayorga, [email protected] | 1Instituto de Química, Universidad Nacional Autónoma de México, Mexico City, México. SN Applied Sciences (2021) 3:566 | https://doi.org/10.1007/s42452-021-04505-8 Vol.:(0123456789) Research Article SN Applied Sciences (2021) 3:566 | https://doi.org/10.1007/s42452-021-04505-8 activation, dimerization activation, transactivation, or relevance of TM7 movement is suggested in arginine- biphasic activation [1]. vasopressin type 2 receptor (V2R) [14] using fuorescence Biased activation of the mu-opioid receptor (μ-OR) spectroscopy; and in β2-adrenergic receptors (β2AR) [15] towards G-protein is relevant for the design of painkillers characterized by 19F-NMR experiments. These studies with reduced detrimental side efects. For example, activa- revealed that movements at TM6 helix are required for tion of β-arrestin signaling pathways is suggested to lead G-protein signaling. In contrast, β-arrestin-biased ligands to tolerance, desensitization, addiction, constipation, and impact the TM7-H8 region. This information could be respiratory suppression [2]. Albeit not fully understood, transferable across class A GPCRs [7, 8, 16, 17] where the these efects seem to be related to β-arrestin recruitment allosteric sodium site is preserved. To note, experimental or receptor internalization. Moreover, the coexistence of information regarding the allosteric sodium site in μ-OR diverse and complex GPCR activation types is an active is scarce [18, 19], but many computational studies have research topic. This complexity is further evidenced by the shown the collapse of the allosteric sodium site in the ability of GPCRs to adopt diferent conformations. Experi- activation and the egress pathways of the sodium ion in mental evidence shows that diferent agonists can stabi- the presence of agonists and modulators [19–21]. These lize these conformations, leading to new conformational studies also suggest the lack of spontaneous release of landscapes [3–5]. This behavior is counterintuitive, based the sodium ion from the bundle which required random on the paradigm that all agonists would conduct to only accelerated molecular dynamics simulations to explore one active conformation [6]. possible outlets from the allosteric site [19]. Besides the biased ligands, experimental evidence This important behavior has motivated the search shows that a conserved allosteric binding site plays a for allosteric modulators in μ-OR. For example, ignavine crucial role in the activation of class A GPCRs that include increases agonist response for DAMGO and has positive μ-OR [7]. Modulation of the GPCRs via this allosteric site is modulatory activity in morphine and DAMGO assays. Fur- related to downstream signaling and physiological efects thermore, ignavine produces an analgesic efect in vivo [8, 9]. The high-resolution (1.8 Å) X-ray crystal structure through opioid response, proved by tail-flick and tail of the adenosine A2A receptor revealed a highly ordered pressure tests [22]. However, the mechanistic modula- cluster of atoms, composed of one sodium ion, water mol- tion transmission in opioid receptors is still unclear. The ecules, and conserved amino acids [8] located on TM2 and positive allosteric modulator (PAM) BMS-986122 of μ-OR, TM3, such as D 2.50 and S 3.39. The importance of an allos- which suppresses the β-arrestin recruitment in conjunc- teric sodium binding site nearby D2.50 has been reviewed tion with agonists [23], can decrease the ability of NaCl to [7–9], particularly on the receptor activation process and modulate the binding afnity of agonists and has been G-protein/arrestin signaling bias. Also, the structure of the proposed to act as an indirect competitor of the allosteric delta-opioid receptor (δ-OR), in the inactive state, showed sodium ion [18]. Additionally, molecular dynamics (MD) an electron density distribution located in an allosteric site simulations of BMS-986122 suggested that residues on in the vicinity of D2.50, corresponding to a sodium ion and TM7 (7.53 and 7.35) are involved in the positive allos- also unveiled N3.35 as an important amino acid in this bind- teric signal transmission [24]. Notably, the structure of ing site [9]. In agreement with these fndings, mutations BMS-986122 contains motifs related to C2-benzyloxy of N3.35 V and N 3.35A decreased and abolished δ-OR agonist herkinorin; this is in agreement with our suggested bind- sodium efect, respectively. Interestingly, in both cases, the ing mode of herkinorin involving modulation of TM7 [25]. δ-OR arrestin-mediated signaling pathway is increased. Comparing the X-ray structures of μ-OR in the active However, both mutations did not afect the canonical sign- [26] and inactive [27] states allows analyzing helical rear- aling, which is mediated by G-proteins. This suggests that rangements related to the activation process. These rear- while the allosteric sodium ion can modulate the recruit- rangements are common to other members of the class ment of β-arrestin, the G-protein–receptor binding only A GPCR superfamily (β-2AR, rhodopsin, M2R) [7, 8, 16], depends on the agonist [9]. Initial experiments showed such as helical movements at TM6 and TM7. Moreover, a dependence on sodium concentration for the afnity changes in water-mediated interactions involving amino of agonists but not for antagonists [10, 11]. Antagonists/ acids inside the receptor’s cavity are observed when the weak partial agonists are also afected by D2.50 mutations, activation is established [26]. The active state of the recep- which become β-arrestin-biased agonist [7]. Other small tor is characterized by an ordered network involving water molecules such as amiloride can compete with the allos- molecules. Notably, careful analysis of the water network teric sodium site [9, 12, 13]. shows that it is functionally relevant, transferring infor- Mutations at D 2.50 and N 3.35 in δ-OR at varied concen- mation from the extracellular side (ligand binding site) tration of Na+ afect recruitment of β-arrestin, most likely through the receptor [26], and reaching the interior of the by affecting the TM7 helical bundle [9]. Similarly, the cell. This last step is characterized by collective movements Vol:.(1234567890) SN Applied Sciences (2021) 3:566 | https://doi.org/10.1007/s42452-021-04505-8 Research Article of intracellular regions of the TMs and loops. This informa- 2 Results tion relates to the exquisite modulation of the receptor that results from ligand binding. The μ-OR biased signaling mechanism, proposed in this Interestingly, herkinorin, a non-nitrogenous μ-OR work, initiated at the orthosteric binding site continues agonist, does not produce β-arrestin2 recruitment and via an allosteric binding site [26], then reaches a hydropho- μ-OR cell internalization, even when the G-protein- bic barrier (framed by DRY and NPxxY conserved motifs) coupled receptor kinase 2 (GRK2) is overexpressed. [34,
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