In Search of GABAA Receptor's Neurosteroid Binding Sites - Journal

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In Search of GABA Receptor´s Neurosteroid Binding Sites Lautaro Damián Alvarez, Adali Pecci, and Dario A Estrin J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01400 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Journal of Medicinal Chemistry

In Search of GABAA Receptor´s Neurosteroid Binding Sites Lautaro D. Alvarez*,†,#, Adali Pecci†,§, and Dario A. Estrin‡,∥

†Universidad

de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Biológica, Ciudad Universitaria, Buenos Aires C1428EGA, Argentina.

‡Universidad

de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química

Inorgánica Analítica y Química Física, Ciudad Universitaria, Buenos Aires C1428EGA, Argentina. #CONICET

– Universidad de Buenos Aires, UMYMFOR, Ciudad Universitaria, Buenos Aires C1428EGA, Argentina.

§CONICET

– Universidad de Buenos Aires, IFIBYNE, Ciudad Universitaria, Buenos Aires C1428EGA, Argentina.

CONICET – Universidad de Buenos Aires, INQUIMAE, Ciudad Universitaria, Buenos Aires

∥

C1428EGA, Argentina.

ACS Paragon Plus Environment

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Abstract. Neurosteroids (NS) are the main modulators of γ-Aminobutyric acid type A Receptors (GABAARs), which are the ligand-gated channels target of the major inhibitory neurotransmitter in vertebrates. As a consequence of their ability to modify inhibitory functions in the brain, NS have high physiological and clinical relevance. Accumulated evidence has strongly suggested that NS binding sites were located in the GABAAR transmembrane domain, however the specific localization of these sites has remained an enigma for decades. Fortunately, recent resolution of GABAARs crystal structures, together with computational strategies applied to investigate the NS binding, have paved the way to rationalizing the molecular basis of NS modulation. This work reviews from a historical perspective the road followed for establishing the GABAAR/NS binding mode, from their initial molecular modeling to the latest findings. Furthermore, a comparative analysis describing the NS binding is provided, plus a preliminary analysis of putative NS sites in other assemblies.


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1. Introduction Neurosteroids (NS) are endogenous steroids acting on the central nervous system (CNS). Biosynthesized in the brain from cholesterol or through conversion of peripherally derived adrenal and gonadal steroids, they are the main modulators of γ-Aminobutyric acid (GABA) action, the major inhibitory neurotransmitter in vertebrates. GABA plays essential roles in virtually all brain physiological functions, such as cognition, memory, and learning, and exerts its action primarily by activating ligand-gated anion channels termed GammaAminobutyric Acid type A Receptors (GABAARs).1–3 This receptor-ligand system is essentially involved in all neuronal circuits, modulating both postsynaptic and presynaptic inhibition. Anxiety, schizophrenia, and epilepsy, among other diseases, have been related to impairments of GABA or NS signaling.4–6 Determining how NS interact with the GABAA receptor is a prerequisite for understanding their physiological and pathophysiological roles in the brain. Furthermore, NS exogenous administration produces clear behavioral manifestations such us anxiolysis, sedation, analgesia, anticonvulsant effects and anesthesia. This makes NS potential pharmacological candidates for the treatment of diverse CNS disorders,7,8 and in this context, multiple new leading compounds are currently evaluated in different clinical studies.9 Thus, a precise understanding of the molecular basis of NS action may redound to better treatments for a variety of diseases, such as epilepsy, in which research into the therapeutic potential of different synthetic NS is showing a considerable resurgence. GABAARs are pentameric membrane-bound proteins belonging to the Cys-loop superfamily of ligand-gated ion channels.10 In mammals, 19 subunits from eight different subtypes (α1-6, β1-3, γ1-3, δ1, ε1, Φ1, π1 and ρ13) can be assembled together in GABAARs. Each subunit consists of three well-defined domains: the extracellular domain (ECD), the transmembrane domain (TMD) formed by four α-helices segments (TM1– TM4), and a cytoplasmic loop of variable length between TM3 and TM4 α-helices (Fig. 1a). TM2 segments of the pentameric receptor constitute the chloride channel that allows the influx of chloride ions from the extracellular to the intracellular compartment. The interface between two adjacent subunits is formed by interactions between TM1 residues from one of them with TM3 residues from the other. TM4 segments are located at the periphery of the channel in close contact with phospholipids of the cell membrane.


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The heteropentameric GABAAR assembly differs throughout the CNS since it depends on the expression abundance of different subunits subtypes, generating a complex structural heterogeneity with different pharmacological profiles.11,12 Despite this, it has been recognized that direct synaptic transmissions are mainly mediated by receptors composed by αβγ subunits in the ratio 2:2:1, with an α-β-γ-α-β arrangement (anticlockwise from the intracellular surface, Fig. 1b). In these receptors, GABA binding pockets localize in α/β extracellular interfaces. Other allosteric modulators, such as benzodiazepines or pyrazoloquinolines, also bind to specific binding sites located in the ECD.13–15 In extrasynaptic GABAARs, γ subunit is replaced by δ subunit, conforming low-efficacy receptors in which the NS binding induces a greater channel opening and nondesensitizing tonic inhibition.16 Foundational studies focusing on the structure-activity relationship helped establish the basis of NS action on the receptor activity. Initially, two discernible effects were observed: i) potentiation of GABA action at nanomolar NS concentrations, and ii) direct activation of GABAAR channel at micromolar NS concentrations. Regarding the steroid structure, conclusive results have shown that both overall bent and planar molecules, such as 3α-hydroxy-5β-pregnan-20-one (pregnanolone, Preg) and 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, Allo) respectively (Fig. 1c), have the ability to modulate the GABAAR activity.17 Moreover, a α-configuration of the 3-OH moiety seems to be essential while 3β-isomers are inactive or behave as antagonists. These findings led to the conclusion that NS modulation occurs through specific GABAARs binding sites. Although accumulated evidence strongly suggested that these sites were located in the TMD, the number and specific localization has remained an enigma for decades, with several models proposed, mainly on the basis of functional properties of point mutated receptors. Recently, both X-rays18,19 and molecular modeling studies20,21 have precisely detailed how endogenous NS specifically bind to GABAARs. These observations have enormously improved the understanding of NS mechanism of action. Briefly, the NS binding sites are located in the subunit interfaces, close to the intracellular side of the TMD. Twelve residues exposed to the membrane constitute the NS binding site, 5 from the TM3 of one subunit, 5 from TM1 and 2 from TM4 of the adjacent subunit (Fig. 2). It has been found that a key structural


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element is the conformation assumed by the indole group of the Trp at TM124 that creates a planar platform in which the steroid lays its planar scaffold maintaining a stacking interaction, while specific protein-ligand hydrogen bonds are formed (Fig. 3). The present work reviews from a historical perspective the road followed from one of the first molecular models of the GABAAR/NS interaction, proposed by Hosie et al. in 2006,22 to the recent findings establishing the NS binding mode (Table 1). The structural determinants of NS binding are correlated with the main experimental results obtained throughout the years. The binding modes of other class of steroids, such as inhibitory sulfated steroids and cholesterol analogues, are not discussed in this work. The main goals of this work are to discuss the historical advances made towards predicting the NS binding mode from the first homology models and to disclose a correct methodology to understand the GABAAR/NS interaction. A comparative analysis among crystals structures and homology models describing the NS binding at α or β homopentameric and α1β2γ2 heteropentameric receptors, and a preliminary analysis of the constitution of putative NS sites in other assemblies are presented. Finally, a speculative model linking NS physiological and pharmacological effects with the NS site occupation is proposed. Improving the understanding of the mechanism of action may enable the precise physiological and pathophysiological roles of NS in the CNS to be established for the first time, as well as providing opportunities for the design of novel drug entities.

2. The first GABAAR/neurosteroid model (2006-2007) Previous to 2006, several studies focusing on the interaction between general anesthetics and GABAARs had been reported. These studies established solid bases to investigate the binding of small molecules to TMD residues. In a seminal work, Mihic et al. investigated the molecular basis for modulation of GABAAR by volatile anaesthetics and alcohols, and in particular identified two specific residues in TM2 and TM3 which are critical for allosteric modulation.23 Later, the propofol binding site was defined using cysteine accessibility experiments,24 while a point mutation in the TM1 affecting anesthetic (etomidate and alphaxalone) modulation


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was characterized.25 Furthermore, the critical role of TMD residues for anesthetic action was proved in vivo by using genetically engineered knock-in mice.26 In 2006, in a context where the presence of unique specific NS binding sites at the GABAAR TMD had been widely accepted, an article by Hosie et al. from Smart’s lab proposed the existence of two discrete NS binding sites in the heteropentameric GABAAR-α1β2γ2, one associated with the potentiation effect and the other with the activation effect.22 Hosie et al. based their hypothesis on results obtained through mutational studies interpreted by using an homology model of the receptor. Briefly, they first found that two residues of the α1TM1, α1Thr236 (TM115) and α1Gln241 (TM120), were essential for the NS action. Then, upon constructing the GABAAR-α1β2γ2 homology model they selected surrounding residues whose localization resulted compatible with the formation of a NS binding site. Finally, by mutating these selected residues, they found that the NS action of these mutants was actually altered, concluding that a potentiation NS site would be formed by α1Gln241 (TM120), α1Asn407 (TM416) and α1Tyr410 (TM419) while a NS activation site would be constituted by α1Thr236 (TM115) and β2Tyr284 (TM37). This idea was further supported by remarkable similarities with the binding mode of similar steroids in their corresponding receptors (i.e progesterone into the progesterone receptor binding pocket).27 In this way, these studies appeared to address a great relevant topic in the field of NS research that was still unresolved, providing a detailed description of both localization and identity of the residues involved in NS binding. Subsequently, this hypothesis was rapidly accepted, guiding the research of a considerable part of the NS community. Almost all studies focusing on GABAAR-NS interaction considered Hosie’s model, which received more than 30 citations/year and more than 450 total citations to date. However, biochemical evidence found in later years proved that the binding sites proposed by Hosie’s model could not be possible. Nowadays, direct evidence led to the conclusion that most of these hypothesis was not consistent with experimental data.18,19 Specifically, their homology model fails in both the overall localization and the assignment of the residues involved in the NS interaction. This is probably due to the fact that the model was constructed starting from a non-optimal template, the nicotinic acetylcholine receptor (AChR) of Torpedo marmotata (PDB ID 1OED). Although those authors did not provide the sequence alignment used to construct the model, a sequence analysis


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indicates that pairwise identities among subunits of AchR (a cation channel) and GABAA (an anion channel) are very low in all cases (less than 20% for TMD residues). Furthermore, multiple alignments showed only one conserved residue when comparing TM1 and TM2, while no conserved residues where found when comparing TM3 and TM4. Differences between M2-M3 loops lengths also challenge the alignment among subunits of both receptors. Sequence alignment is critical in order to construct an homology model, especially for systems formed by parallel α-helices. Insertions or deletions have a large impact on residue positions relative to the membrane and with respect to other receptor residues. Without a reliable model, it is difficult to exclude the hypothesis that mutated residues are not important for binding but, for example, are crucial for transducing binding into a conformational change. As the sequence alignment of AchR and GABAA TMDs is not trivial, the construction of a good GABAAR model based on the coordinates of PDB ID 1OED is extremely challenging and probably there was no better template to use back in 2006. These authors obtained the molecular model for GABAAR/3α,21-dihydroxy-5αpregnan-20-one (THDOC, Fig. 1c) by manual docking instead of more general docking approaches. In spite of the mentioned limitations of the homology model by Hosie et al., this was, for many years, one of the most influential studies considered by researchers exploring GABAAR-NS interactions.

3. Other neurosteroid binding models arising from mutational studies (2008-2012) In 2008, two years after the article by Hosie et al., Akk et al. reported a more detailed kinetic and pharmacological characterization of αTM1 residues using a combination of whole-cell and single-channel recordings on several receptor mutants.28 These authors confirmed the crucial role of α1Gln241 (TM120) and found two more α1TM1 residues, α1Ser240 (TM119) and α1Trp245 (TM124), whose point mutations interfere with the ability of NS to modulate the GABAARs. They only provided an homology model of the α1 subunit by using the molecular structure of AchR of Torpedo marmotata (PDB ID 2BG9) as a template. Despite the Cα atoms of Gln241, Ser240 and Trp245 being apparently well localized, the side chain conformation assigned to Trp245 was erroneous, as has been demonstrated by more recent studies.18,19


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In 2009, a new study from Smart’s lab comparing the activity of several GABAAR-αβγ mutants, concluded that NS potentiation is universally dependent on the presence of Gln at αTM120.29 In fact, these authors inferred that the potentiation NS binding site is highly conserved, and they proposed an alternative model of GABAARα1β2γ2, based as well on the coordinates of the TMD of the Torpedo marmotata AchR receptor (PDB ID 1OED), but constructed throughout a different modeling algorithm. These authors stated relevant differences between this new model and the original homology model, particularly in the coordinates of the residues conforming the NS sites. Nevertheless, they concluded that, in both models, the basic principle they had established for NS binding sites appeared to be preserved, namely that the potentiation site is associated only with the α subunit, and that the activation site is localized in the interface between α and β subunits. This has later been proved to be erroneous, and thus, it can be concluded that AchR appears to be an inappropriate template for modeling of the GABAAR. Three years later, the significance of the α1TM1 region was also highlighted by Bracamontes et al., who demonstrated that this TM1 region can be embedded in other GABAAR subunits (β2 or γ2 subunits) conferring potentiation activity to defective α1 GABAARs.30 Therefore, α1TM1 per se appeared to be sufficient for ensuring NS binding. Nonetheless, TM3 residues had also been found to be determinant for NS response. In 2011, Williams demonstrated that a TM317 mutant, α1Ser299Cys, exhibited extreme and unusual sensitivity to NS.31 Although molecular representations were not provided, the localization of α1Ser299 was indicated in a helical wheel scheme. These projections were also used by Li et al. to compare the putative localization of etomidate (another TMD allosteric modulator of GABAARs) and NS sites, concluding that their experimental results were inconsistent with Hoise’s model.32

4. A model based on the use of a photolabeled neurosteroid (2012) In 2012, Chen et al. used a different approach to identify the localization of the NS binding sites.33 These authors used a photolabeled NS analog, 6-aziprogesterone (6-AziP), to label β3 homopentamers. In a previous report, a photoreactive analog of etomidate had been successfully used to identify directly the TMD amino acids


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contributing to a binding site.34 Functional assays with 6-AziP were consistent with a single class of NS binding site, while photolabeling of β3 subunits was stereoselective ([3H]6-AziP provoked substantially greater labeling than its 3β diastereomer). High-resolution mass spectrometric analysis of purified 6-AziP-labeled β3 subunits identified a single photolabeled residue in the TM3 domain: β3Phe301 (TM324). Remarkably, no other sites of photoincorporation were observed. Nevertheless, as homopentamers are not found in native tissues, these conclusions should be taken with caution. Furthermore, Chen et al. constructed an homology model of β3 homopentamer by using the X-ray structure coordinates of another inhibitory anion-selective channel, the C. elegans glutamate-gated chloride channel (GluCl, PDB ID 3RHW).33 The PDB ID 3RHW structure, representing the first 3D structure of an inhibitory anionselective channel, had been resolved a year earlier.35 GABAAR-β3 and Glu-Cl share 47% TMD identity, with various residues conserved in the four TMD α-helices. In this sense, the Glu-Cl template appeared to be a better template to obtain a more reliable homology model of GABAAR-β3 TMD, since the position of each TMD residue could be assigned unequivocally. This new model showed β3Phe301 residues close to the intracellular region, with the side chain exposed directly to the membrane. Upon determining the position of residues located less than 9 Å from the β3Phe301 (TM324), the authors concluded that the NS binding site should be placed on the TM3 surface, either at the TM3-TM4 interface or at the interfaces between TM3 and TM1 or TM4 of the adjacent subunit. Thus, this model was able to identify the overall localization of NS binding sites. Furthermore, the pillar Trp at TM124, and some other residues conforming the NS binding sites, were modeled in the correct conformation. Although Chen et al. did not use docking schemes to investigate the putative binding of NS at the β3 homopentamer model, reasonable predictions could have been obtained for the NS binding mode.

5. The crystal structure of GABAAR-β3 and molecular modeling (2014-2015) In 2014, Miller et al. reported the first crystal structure of a GABAAR: a β3 homopentamer bound to benzamidine at the ECD, but without ligands at the TMD (PDB ID 4COF).36 Surprisingly, although these authors described exhaustively the homopentameric structure and analyzed the putative binding sites for several


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drugs, the NS binding sites were neither examined nor discussed nor contrasted to the prevailing Hosie’s model. Even more noteworthy is that the simple analysis of the GABAAR-β3 TMD surface clearly shows the presence of a deep surface cavity near β3Phe301 (TM324), mainly delimited by the β3Trp241 (TM124), with an adequate dimension to accommodate a steroid molecule. This auspicious observation led to a primary objective: to computationally demonstrate that these cavities effectively constitute the NS binding sites at GABAAR-β3. For this purpose, in 2005, a series of well validated molecular modeling tools was applied to detect surface cavities, to dock the NS within these cavities and to analyze the binding stability through Molecular Dynamics (MD) simulations of the GABAAR-β3/NS complexes immersed in a membrane model.20 The computational data was conclusive: five identical NS binding sites in the GABAAR-β3 (one per each subunit interface) have the ability to specifically recognize both bent 5β and planar 5α steroids. This model explains satisfactorily the main experimental observations accumulated during the previous years. Firstly, the unique residue that was photolabeled by 6-AziP, β3Phe301 (TM324), is actually observed as part of the NS binding site (Fig. 3b). Although the distance between the C6 of the steroid –where the reactive group is introduced- and the Phe301 is too large to envisage a direct interaction, the reaction between the photoreactive group and the aromatic ring could take place during the ligand binding or unbinding processes. Secondly, the stereospecifity in the C3 atom is clearly explained by the relative orientation assumed by the steroid and the Trp at TM124 (one of the residues that had been found to be essential for NS modulation28). Only steroids with a 3α-hydroxyl moiety have the ability to form a hydrogen bond with the nitrogen atom of the Trp indole group. In fact, MD simulations showed an unstable binding mode for the 3β isomers.20 Furthermore, the proposed NS binding mode also explains the results obtained by Krishnan et al. regarding the activity of enantiomer pairs containing bulky substituents at either C7 or C11.37 These authors showed that 11β-Obn-substituted steroids and 7α-Obn-substituted ent-steroids conserved NS activities (which was abrogated by the α1Q241L mutation in both cases, indicating that they act at the same sites), but 7β-Obn-substituted steroids and 11α-Obn-substituted ent-steroids have strongly diminished activity. The predicted NS sites provide a direct explanation of these findings. Orientations of active analogues are inverted relative to each other, with


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the 11β- (natural) or 7α- (ent-steroids) substituents exposed to the membrane, without affecting the binding. On the contrary, the binding is impeded for the inactive steroids due to the crash between the bulky substituents and protein atoms. Both Chen et al.33 and Miller et al.36 have used β3 homopentamers as a simplified model of GABAAR action. However, β3 homopentamers have not been identified as discrete populations in the CNS and they exhibit distinct functionality compared to heteropentameric α1β2γ2 receptors in artificial systems. Although GABAARβ3 efficiently forms a functional channel that can be modulated by some general anesthetics, such as barbiturates, it is insensitive to GABA action.38 Furthermore, Allo treatment of HEK293 cells expressing the β3 homopentameric receptor causes a small enhancement of propofol-activated currents and strongly inhibits the [35S]TBPS binding, indicating that upon binding, this steroid actually produces structural changes; however it does not activate discernible currents in electrophysiological assays.38 In this context, it is not trivial to assign the GABAAR-β3 NS binding sites to potentiation or activation functional effects, mainly due to the fact that these effects have not been characterized in β3 homopentamers (see below).

6. Crystal structures of GABAAR-α/neurosteroid complexes (2017-2018) In 2017, two years after the computational model of GABAAR-β3-NS interaction was proposed,20 two independent groups simultaneously reported crystal structures of GABAAR/NS complexes. Since no crystallizable α homopentameric assemblies could be obtained, these two groups constructed distinct GABAAR chimeras containing α TMDs. Miller et al. generated a chimeric receptor by fusing the α5TMD to the β3ECD, and proved that this construction forms a functional gating unit.18 X-ray structures of this chimera were determined in the absence and presence of Preg (PDB ID 5OJM and 5O8F, respectively). On the other side, Laverty et al. developed a new ‘prokaryotic–eukaryotic’ chimera, which includes the ECD of the prokaryotic homolog GLIC from Gloeobacter violaceus with GABAAR α1TMD.19 They also proved that this chimera responds to NS and determined their structure bound to THDOC (PDB ID 5OSB). More recently, in 2018, a third GABAAR chimera was generated by fusing the TMD of human GABAAR α1 to the ECD of ELIC, a


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prokaryotic pLGIC from Erwinia chrysanthemi, and the authors determined its crystal structure bound to a synthetic derivative of Allo, the alphaxalone, (PDB ID 6CDU).39 Notably, the localization of the NS binding sites and the overall steroid orientation observed in the αTMDs of the three crystal structures of GABAAR chimeras (PDB ID 5O8F, 5OSB and 6CDU) fully coincided with those predicted by molecular modeling on GABAAR-β3 TMD (PDB ID 4COF).20 Similarly to the β3 heteropentamer, the αTMDs of the crystal structures present five identical sites in the TM1-TM3 subunit interfaces where the steroid is specifically recognized (Fig. 3a). Comparison between apo and holo GABAAR-α5 TMD reveals very similar structures, with NS binding residues mainly localized at the same positions. Thus, no large conformational changes are required for NS binding. Moreover, the essential role in NS binding of Gln at TM120 position is clearly evidenced from the crystal structures: α1Gln241 and α5Gln245 orientate their side chain towards the steroid A-ring forming a hydrogen bond with the 3-OH moiety.

7. A new GABAAR-α1β2γ2/neurosteroid complex homology model (2018) In view of the successful application of molecular modeling to predict the NS binding in GABAAR-β3, a similar computational strategy was applied to investigate the NS binding to α1β2γ2 heteropentamer receptors,21 which is the most abundant and physiologically relevant GABAAR assembly in humans. Initially, an homology model of the GABAAR-α1β2γ2 TMD was constructed using the β3 homopentamer crystal structure coordinates (PDB ID 4COF) as template. The conservation of several TMD residues allowed a direct alignment of α1, β2, β3 and γ2 subunits (Fig. 2), which instilled confidence in the created model. In contrast to homopentamers, four different NS cavities (i.e. where at least one residue differs) can be distinguished in the GABAAR-α1β2γ2. The inspection of the NS binding sites revealed that only a few residues were changed respect to β3 homopentamer structures.21 The overall shape of the binding sites is essentially conserved in the four different NS cavities of the heteropentameric receptor, all of them maintaining an adequate volume to bind steroids. A similar docking/MD simulation approach was then applied to evaluate the NS binding


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at the heteropentameric cavities. Again, molecular modeling was conclusive: all of GABAAR-α1β2γ2 cavities conserved the ability to specifically recognize NS, producing very stable binding modes, similar to those depicted above for homopentameric receptors. Early in 2018, three cryo-electron microscopy structures of heteropentameric GABA R-αβγ were reported: A

GABAAR-α1β2γ2 in complex with GABA and flumazenil (PDB ID 6D6U and 6D6T),40 GABAAR-α1β1γ2 in complex with GABA (PDB ID 6DW1)41 and GABAAR-α1β3γ2 in complex with GABA and a nanobody that acts as a novel positive allosteric modulator.42 Although these studies provide excellent templates for understanding GABA and benzodiazepines action, no relevant information on the NS binding can be extracted. Since these structures were obtained in the presence of detergents and TMDs deviate from the five-fold symmetry, the authors interpret these conformational states with caution. The conformations of the Trp residue at TM120 are similar to those observed in the crystal structures, but the overall shape of NS binding sites is largely deformed in the majority of subunit interfaces, suggesting that these TMD structures may represent non-native conformations. The reliability of these TMD conformations is therefore questionable, and further analysis is required to determine the relevance of these TMD conformations for the physiological action of the receptor.

8. Comparison of neurosteroid binding mode in homopentameric and heteropentameric GABAARs/NS complexes Computational tools predicted accurately both NS sites localization and overall binding mode. The relative orientations between the steroid and the indole group of the Trp at TM124, and between the steroid and the membrane (axis z) are very similar in experimental and modeled structures. The analysis of all GABAAR/Preg structures revealed six different NS binding cavities that can be distinguished from a total of 15 cavities: one in the GABAAR-α5, another in the GABAAR-β3 and four in the GABAAR-α1β2γ2. Overall, two different binding modes can be observed, mainly determined by the identity of the residue at TM120. When a Gln occupies this position, an interaction with the NS 3-OH group is firmly established (Mode 1). Instead, when the residue at TM120 is a Trp, the 3-OH group interacts with the backbone of the TM45 residue (Mode 2). Predictions for the


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interaction free energy using MD based a continuum solvent MM-PBSA methodology calculations performed on the GABAAR-α1β2γ2 revealed very similar Preg affinity between Mode 1 and Mode 2.21 Thus, NS binding appears equally favored in both binding modes. The Mode 1 sites can be convincingly assigned to the potentiation sites of the GABAAR-α1β2γ2, but the role of Mode 2 sites in α1β2γ2 heteropentamers is not clear (see below). The NS binding at Mode 2 sites, although insufficient to directly activate the channel, appears to have an impact on distant regions of the receptor structure in β3 homopentamers. The main difference in Mode 1 binding, when comparing crystal and modeled structures, resides in the distance between the NS 3-OH group and the nitrogen atom of the indole of the pillar Trp at TM124. This distance is larger than 3 Å in the crystal structures, suggesting that a hydrogen bond between both polar moieties is not formed. On the other hand, MD simulations do reveal a persistent interaction between these atoms in all analyzed systems. Whether the lack of this interaction is an artifact originating from the crystal structure or a particularity of TMD formed exclusively by α subunits, this remains unclear. However, preliminary MD simulations of αTMD bound to Preg show that this system promptly evolves to establish this interaction (unpublished results). Nevertheless, the role of plasma membrane on the kinetic and steady-state properties of NS-receptor interactions should be also contemplated.43 Inspecting the GABAAR-α5/Preg complex (PDB ID 5O8F), Miller et al.18 have suggested a putative hydrogen bond between the steroid C20 ketone group and the hydroxyl group of the α5Thr309 (TM324). However, a more detailed analysis of this crystal structure reveals that the oxygen atom of the hydroxyl group of α5Thr309 (TM324) is located closer to the oxygen backbone atom of the α5Ile305 (TM320) than to the C20carbonyl oxygen atom (Fig. 3a). This observation suggests that the α5Thr309 (TM324) side chain forms a protein-protein interaction rather a protein-ligand hydrogen bond. The above conclusion is consistent with the MD results obtained with the GABAAR-α1β2γ2/Preg complex (Fig. 3c), where no polar interactions appear to establish between the steroid side chain and receptor residues. Furthermore, there are no NS cavities with polar amino acids at both extremes of the steroid in α1β2γ2 heteropentamers.21


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The comparison between complexes with planar steroid bound, GABAAR-α1/THDOC (PDB ID 5OSB), GABAAR-α1/Alphaxalone (PDB ID 6CDU) and GABAAR-α1β2γ2/Allo, also reveals similarity between crystal and modeled structures (Fig. 4). Steroids are positioned parallel to the Trp side chain of TM124 maintaining a stacking interaction. Again, no hydrogen bond between Trp at TM124 and the NS 3-OH group is evidenced from the analysis of the crystal structure (Fig. 4a), although this interaction was established in the MD simulations (Fig. 4b,c).20,21 Similarly to Preg, THDOC and Allo form hydrogen bonds with the Gln at TM120 in Mode 1 sites. However, in Mode 2 sites the hydrogen bond acceptor of the NS 3-OH group differs between these isomers. While the Preg 3-OH group forms a hydrogen bond with the TM45 oxygen backbone atom, Allo 3-OH group acquires a different orientation by contacting the TM120 oxygen backbone atom. The GABAAR-α1/THDOC crystal structure reveals that the ligand C21-OH group is pointing towards the membrane, without contacting receptor atoms. However, structure-activity relationship studies have revealed that the pharmacophore model for the positive modulation of the GABAAR contains a hydrogen bond-accepting group in a pseudoequatorial orientation at the 17β of the steroid.17 The reason why this orientation is required can now be understood, but further studies will be necessary to fully recognize the role of the steroid side chain. Again, it should be taken into account that GABAAR-α1β2γ2 Mode 1 cavities do not exhibit polar residues at position TM324. MD simulations of homopentameric and heteropentameric GABAA-Rs immersed in an explicit membrane model allowed studying the behavior of NS cavities in the absence of NS.20,21 The MD results revealed two alternative states for the free NS cavities: expanded or collapsed. In the expanded state, lipid molecules fill the cavities forming an extensive contact with the hydrophobic residues, especially with the planar surface of the Trp at TM120. When the lipids are removed, the NS can be docked properly into these cavities, in agreement with the conclusion extracted from the analysis of apo and holo GABAAR-α5 TMD structures. Contrary to this, in the collapsed state, receptor residues move to form protein-protein interactions, excluding lipid molecules from the cavity. Further investigation is needed to determine whether collapsed cavities are, or not, an artifact


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derived by the force field limitations and/or by incomplete sampling in the MD simulation. Nevertheless, experimental and modeling results convincingly suggest that GABAARs have pre-formed NS cavities.

9. Neurosteroid binding sites at other subunits assemblies. Considering that in mammal GABAARs there are 19 subunits from eight different subtypes that can be assembled together, the large number of putative GABAAR arrangements implies, a priori, a great structural heterogeneity among subunit interfaces to where NS can bind. However, the high level of conservation of the majority of residues participating in the NS binding suggests that NS cavities can be generated in practically any subunit combination. First and foremost, the residue that mostly interacts with the steroid determining the binding specificity, i.e. the Trp at TM124, is conserved in all 19 GABAA subunits. Therefore, any subunit interface has a Trp exposed to the membrane with the potential ability to interact with NS. Other human membrane receptors belonging to the Cys-loop superfamily of ligand-gated ion channels present a Trp residue at this position, and are also preceded by a conserved Val-Ser-Phe sequence. From these, it was observed that Allo analogues have the ability to potentiate glycine receptors,44,45 although little is known regarding the localization of NS binding sites on these receptors. AchRs and serotonin 5-HT3 receptors also have a Val-Ser-Phe-Trp sequence in the TM1, but NS effects have still not been investigated in detail for these receptors. Thus, it would be interesting to analyze the existence and functionality of cavities around TM124 in these receptors. Also, TM3 and TM4 residues from the adjacent subunit seem to have a relevant structural role in GABAARs. The triad of residues TM124-TM327-TM45 appears to be core in forming NS cavities. The polar interaction formed between the Tyr at TM327 (present in all subtypes except  and ) and the Arg at TM45 (present in all subtypes except ) could play an essential role in anchoring the conformation of the Trp at TM124. Both crystal structures and MD simulations suggest that a cation- stacking is formed between the guanidinium and the indole groups. Whether the sole presence of these residues is sufficient to create a NS binding site should be further investigated. Residues located at positions TM121 (Val) and TM123 (Phe) are also completely, or almost


16

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completely, conserved among subunits. In the TM3 domain, position TM316 (Phe) is completely conserved while the residue at position TM323 is generally a residue with small side chain (Ala or Gly), except in the Φ subunit (Val). Other NS binding residues are more variable throughout the GABAARs family of receptors. For example, a hydrophobic residue (Ile or Val) is generally found at TM117, but some subtypes present a Met at this position. Substitution of hydrophobic residues by Met is also produced in TM320 of γ1 and γ3 subtypes. The change in hydrophobicity caused by the introduction of a sulfur atom in the side chain of TM320 could have an impact in NS molecular recognition. The most variable position of the NS binding site is that of TM324, where very chemically different residues can be observed: Thr, Ala, Phe, Leu, Val and Tyr. To study how these variations affect the recognition of C17 NS substituents would be very valuable for the rational design of potentially selective NS. The extrasynaptic GABAARs containing  subunits are a very important case. In these receptors, NS exhibit greater sensitivity,46 but the pharmacophore model developed upon functional screening of a vast library of NS indicates high similarity with that obtained for GABAAR-αβγ.47 What are the implications of the replacement of Tyr by Phe at position TM327 (i.e. the deletion of the hydroxyl moiety that confers ability to interact with the Arg at TM45) on the cavity structure and the NS binding? This and other possible variations should be thoroughly analyzed to further understand the molecular details of extrasynaptic NS action.

10. A hypothesis regarding the activation and potentiation effects of neurosteroids in α1β2γ2 receptors. In 2006, the ability to distinguish two types of NS effects in heteropentameric receptors led to the idea that two discrete sites should exist for potentiation and activation responses.22 Modeling of the GABAAR-α1β2γ2 has led to four different classes of NS binding cavities with two overall different binding modes. However, these two overall binding modes cannot be directly associated with a particular NS effect.


17


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A simple hypothesis explaining the activation/potentiation effects could be based on the occupation degree of GABAAR cavities (Fig. 5). Evidently, this proposal does not take into account other mechanisms that could affect channel activity, such as possible unspecific actions of NS through the modification of membrane physical properties that occur upon steroid embedding in the lipid environment. We propose that in presence of GABA, low concentration of NS may bind to either of the NS cavities with similar affinities. In Mode 1 cavities, the interaction between NS and the Gln residue at TM120 initiates a mechanism that derives in GABA potentiation. It has been shown that NS potentiation in α1β2γ2 receptors does not require steroid interaction with a particular subunit of the GABAAR, nor with a subunit that also binds GABA.30 Evaluating concatenated subunits, it was found that Mode 1 sites are similar in terms of steroid recognition and functional effects.48,49 Moreover, the NS presence at either Mode 1 site can induce potentiation, but both sites are involved in producing full effects. Although further studies could provide more precise information, changes observed by MD in the TM1-TM4 interaction that occur upon steroid binding could be associated to the allosteric communication between NS and GABA.21As part of this hypothesis, we propose that GABA action is not affected by the binding of NS molecules to Mode 2 cavities. In the absence of GABA, alternative binding of NS to Mode 1 or Mode 2 cavities does not produce significant effects on the channel state, but at high NS concentrations, all cavities (Modes 1 and 2) would be occupied, provoking a direct channel activation. The above hypothesis should be contrasted with activity results from receptors formed by concatenated subunits that can specifically inhibit the NS binding to Mode 2 cavities. Nevertheless, another hypothesis to evaluate the different NS effects would assign the potentiation effects to Mode 1 binding, with no functional effects in Mode 2 cavities, and the activation effects produced by other different events, such as the binding to other regions or the unspecific action on membrane dynamics.

11. Final Remarks When crystal structures are not available, reliance on homology models is mandatory for the rationalization of the ligand-receptor interaction at a molecular level. A low quality model will inevitably lead to erroneous


18

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interpretation of experimental data, while a good quality model has potentially both explanatory and predictive capabilities. The molecular modeling of the GABAAR/NS binding is a successful example. Two years before the resolution of the GABAAR/NS crystal structures, docking and MD simulation methods were capable of predicting the molecular determinants of the GABAAR/NS binding. Furthermore, the model derived from GABAAR-β3 structure was able to explain the essential role of Gln at position TM120 in α subunits, even when those crystal structures had yet not been resolved. Thus, this receptor-ligand system represents a successful application of molecular modeling. In a similar fashion, this strategy could be accurately extended to the study of NS binding to other GABAAR subtype combinations. The resolution of the NS binding enigma largely improves the understanding of NS action. Many opportunities now become available in the search for more effective NS ligands. The same can be said for the study of the allosteric mechanisms involving GABA, NS and channel action. In this case, a complete model of GABAAR-α1β2γ2 including ECD and TMD should be necessary and the dynamical behavior of the receptor in the absence or presence of NS and/or GABA should be analyzed using MD simulations. For this purpose, the GABAAR-β3 appears as a more adequate template than the artificial α crystallized chimeras, in which unrealistic contacts in the ECM-TMD interface occur. Furthermore, the role of the intracellular loop should not be ignored. The deciphering of the GABAAR/NS binding should allow for an exhaustive revision of the structure-activity evidence accumulated in the last decades for both endogenous and synthetic NS analogs. In this sense, the binding mode of more elaborated clinical candidates – having larger steroid side chains, such as SAGE-21750 – should be analyzed in detail since these synthetic analogs might not exactly bind at the same sites of endogenous compounds. The availability of a consolidated and reliable GABAAR/NS model should provide valuable information to explain the influence of steroid structure on the pharmacological activity, allowing a rational structure-based design of new NS analogs. GABAARs are molecular machines with diverse structural arrangements that are involved in practically all neuronal circuits. As precise molecular knowledge of these proteins is improved, new strategies to control their functional properties could be developed. Thus, the


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elucidation of NS binding determinants opens a new era to achieve this objective, providing opportunities for novel therapeutic approaches.

Acknowledgement We thank Dr. Rodolfo R. Biekofsky for critical reading of the manuscript.

Abbreviation Used NS, neurosteroids; GABA, γ-Aminobutyric acid; GABAARs, Gamma-Aminobutyric Acid type A Receptors; ECD, Extracellular domain; TMD, Transmembrane domain; ICD, Intracellular domain; Preg, 3α-hydroxy-5βpregnan-20-one; Allo, 3α-hydroxy-5α-pregnan-20-one; AChR, nicotinic acetylcholine receptor; THDOC, 3α,21dihydroxy-5α-pregnan-20-one; 6-AziP, 6-Aziprogesterone; Glu-Cl, glutamate-gated chloride channel; MD, molecular dynamics; MM-PBSA: Poisson–Boltzmann surface area continuum solvation method.

Author Information Corresponding Author: *Email: [email protected] Notes: The authors declare no competing financial interest Biographies: Lautaro A. Alvarez received his degree in Chemistry from the School of Sciences of the University of Buenos Aires in 2005 and his PhD from the same University in 2009. He is currently Lecturer at the School of Sciences, University of Buenos Aires and researcher at the Unity of Microanalysis and Physical Methods in Organic (UMYMFOR-CONICET-UBA). His main research interest concerns the study of the molecular basis of steroid


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actions in nuclear receptors and pentameric ligand-gated channels, with a focus in the application of molecular modeling tools to investigate ligand binding and allosteric mechanisms.

Adali Pecci received her degree in Chemistry from the School of Sciences of the University of Buenos Aires in 1987 and his PhD from the same University in 1994. Later on, she got a postdoctoral position at Institut für Molekular Biologie und Tumorforschung, Philipps Universität, Marburg, Germany. She is currently Associate Professor at the School of Sciences, University of Buenos Aires and researcher at the Institute of Physiology, Molecular Biology and Neurosciences (IFIBYNE-CONICET). Her research is focused in the molecular mechanisms of steroids and steroid receptors to control gene expression regulation during cell proliferation and tumorigenesis.

Dario A. Estrin received his degree in Chemistry from the School of Sciences of the University of Buenos Aires in 1986 and his PhD from La Plata University in 1989. Later on, he got postdoctoral positions at Ohio State University and at the Center of Advanced Research Studies of Sardinia (CRS4). He is currently full Professor at the School of Sciences, University of Buenos Aires and researcher at the Institute of Physical Chemistry of Materials, Environment and Energy (INQUIMAE-CONICET). His contributions have been recognized with several national and international awards, including the Guggenheim Fellowship. His research is aimed at the development and application of computer simulation schemes to investigate reactivity, dynamics, and spectroscopy of biomolecules, with a focus on multi scale QM-MM methods.

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Legends.

Fig. 1. a) Lateral view of the GABAAR-α1β2γ2/progesterone model21 showing the extra cellular (ECD) and the transmembrane (TMD) domains. b) View from the intracellular space showing the localization of the four TM αhelices (TM1-TM4). The intracellular domain (ICD) is omitted since no crystal structure has been determined for this region. Pregnanolone is showed in black. c) Structures of NS that interact with GABAARs. Numbers of relevant carbon atoms are indicated. 3D structures of endogenous NS pregnanolone and allopregnanolone are showed at bottom. Fig. 2. a) Sequence alignment of TM1, TM3 and TM4 residues of the 19 human GABAAR subunits. Residues forming the NS binding sites are highlighted. b) Schematical representation of the NS binding cavity with pregnanolone bound. Cα atoms of the 12 residues involved in NS binding are showed as orange spheres. Fig. 3. Schematic representation of pregnanolone binding modes in GABAAR-α5 (a, PDB ID 5O8F), in GABAAR-β3 (b, Molecular modeling)20 and in GABAAR-α1β2γ2 (c, Molecular modeling).21 A representation of the TMD assembly where the binding mode at each TM1-TM3 interface is showed (blue triangles for Mode 1 and red squares for Mode 2). Fig. 4. a) Schematic representation of the THDOC binding mode in GABAAR-α5 (PDB ID 5OSB). b) Schematic representation of the allopregnanolone binding mode in GABAAR-β3 (Molecular modeling).20 c) Schematic representation of the allopregnanolone binding mode in GABAAR-α1β2γ2 (Molecular modeling).21 Fig. 5. Hypothesis of GABAAR-α1β2γ2 NS cavities occupation degree and potentiation/activation actions (blue triangles for Mode 1 and red squares for Mode 2).


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Table 1. Most relevant studies investigating the NS binding at GABAARs year

structure

source

template

observations

ref

2006

GABAARα1β2γ2/THDOC

Homology 3D model

AchR (PDB ID 1OED)

First model of the GABAAR/NS interaction used to explain experimental results from mutational studies. Erroneous prediction of NS binding site localization.

22

2008

GABAA α1

Homology 3D model

AchR (PDB ID 2BG9)

Model of the α1 subunit alone, showing the essential TM1 residues.

28

2012

GABAAR-β3

Homology 3D model

Glu-Cl (PDB ID 3RHW)

Model of the β3 homopentamer showing the position of the photolabelled Phe301 residue. Although NS docking was not evaluated, this model appears to be able to predict NS binding.

33

2014

GABAAR-β3

Crystal structure: PDB ID 4COF

First X-rays structure of a GABAAR. NS sites were not inspected.

36

2015

GABAAR-β3/Preg

Molecular modeling

Computational tools (cavity detection, docking and MD simulations) applied on the PDB ID 4COF. Successful prediction of both NS sites localization and NS orientation. First NS binding site identified for a GABAARs.

20

GABAAR-β3/Allo

GABAAR-β3 (PDB ID 4COF)

2017

GABAAR-α5/Preg

Crystal structure: PDB ID 5O8F

-

X-rays structure of a GABAAR chimera in complex with pregnanolone.

18

2017

GABAAR-α1/THDOC

Crystal structure: PDB ID 5OSB

-

X-rays structure of a GABAAR chimera in complex with THDOC.

19

2018

GABAAR-α1β2γ2/Preg GABAAR-α1β2γ2/Allo

Homology 3D model plus molecular modeling

Model of the heteropentameric α1β2γ2 receptor plus the application of computational tools (docking and MD simulations). Successful prediction of NS orientation and the role of α1Gln241.

21

2018

GABAARα1/Alphaxalone

Crystal structure: PDB ID 6CDU

X-rays structure of a GABAAR chimera in complex with alphaxalone.

39

GABAAR-β3 (PDB ID 4COF)

-


29


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Figure 1


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Figure 3

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Figure 4

Figure 5


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In Search of GABAA Receptor's Neurosteroid Binding Sites - Journal

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