Structure and mechanism of the GABA B VFTs

The GABAB-R is an obligate heterodimer comprised of the GABAB1a/b and GABAB2 subunits (Calver et al., 2000; Geng et al., 2013) where each subunit contains an extracellular domain connected to a heptahelical transmembrane domain by a linker region (Fig. 6). GABAB1a and GABAB1b are isoforms encoded by the same gene GABBR1, and structurally they only differ in the N-terminal region with the presence of a sushi domain on the GABAB1a subunit (Biermann et al., 2010). The sushi domain is reported to function as an intracellular sorting signal responsible for trafficking this isoform into axons (Biermann et al., 2010) and has not been implicated to affect the pharmacology or kinetics in heterologous cells (Benke, 2012; Gassmann and Bettler, 2012).

Radiolabeled ligand- and site-directed mutagenesis studies, and later X-ray crystal structures have shown that in contrast to the mGluRs, only the VFT of the GABAB1a/b subunit and not the VFT of GABAB2 contains a binding site for the endogenous agonist GABA (Geng et al., 2013;

Jones et al., 1998; Kniazeff et al., 2002; Urwyler et al., 2005). In addition, sequence analysis show that none of the residues implicated in ligand binding in GABAB1a/b are conserved in GABAB2 VFT (Geng et al., 2012). In the active closed state of the GABAB1a VFT, the GABAB2

VFT remains in an open inactive state (Geng et al., 2013). Also, binding studies with recombinant receptor mutants showed that the VFT of GABAB1a/b is functional in the absence of the GABAB2 VFT, but with reduced agonist affinities (Liu et al., 2004; Nomura et al., 2008).

In addition to increasing agonist affinity, the GABAB2 VFT is suggested to impact receptor activation by promoting signal transduction from the extracellular side to the intracellular site

contributing to increased agonist efficacy (Liu et al., 2004; Nomura et al., 2008). The sequence identity between GABAB1a/b and GABAB2 VFTs is 33% (Frangaj and Fan, 2017).

There are in total nine available X-ray crystal structures of the GABAB-R VFT dimer in the PDB (Geng et al., 2013), but the complete 3D structure that includes all receptor domains has not been solved. The structures show that the VFT heterodimer is formed by non-covalent interactions between the GABAB1a/b and GABAB2 (Fig. 8) and each VFT contains two distinct domains, the N-terminal Lobe 1 (LB1) and the C-terminal Lobe 2 (LB2) (Figs. 6 and 8). The GABAB1a/b and GABAB2 VFTs are similar in sequence length (approximately 400 residues), and the LB1 and LB2 of GABAB1a/b are structurally similar to the corresponding lobes of GABAB2 (Fig. 8) (Geng et al., 2013). The X-ray structures show that the LB1 of GABAB1a/b VFT interacts with the LB1 of GABAB2 VFT both in the active and inactive VFT states. The interactions between LB1-LB1 in the VFTs are fully facilitated by non-covalent interactions which involves patches of hydrophobic interactions, hydrogen bonds and a salt bridge (Geng et al., 2013). The hydrophobic interactions are mainly facilitated by three conserved tyrosine residues that form stacking interactions at the LB1-LB1 interface (Fig. 8).

Upon receptor activation, large conformational changes causes the LB2 domains of GABAB1a/b and GABAB2 to form an additional large non-covalent heterodimer interface (Geng et al., 2013) (Fig. 8). The LB2-LB2 interface is facilitated by a rich hydrogen bond network including the key residues Thr198, Glu201 and Ser225 of GABAB1a/b LB2 and Asp204, Gln206, Asn213 and Ser233 of GABAB2 VFT LB2 (Fig. 8). A site-directed mutagenesis study of the interfacial residues observed in the activated VFT dimer, showed that the conserved tyrosine residues were important for agonist dependent Gi-protein activity and GABA-induced GIRK currents (Geng et al., 2012; Rondard et al., 2008). The mutations also decreased the GABA induced stimulation of [³⁵S]GTP-γS binding, but had no effect on the GABA affinity (Geng et al., 2012).

Figure 8 – The heterodimeric extracellular GABAB-R VFTs in the active state with amino acids important for Lobe1-Lobe1 and Lobe2-Lobe2 interactions displayed. Blue – GABAB1a/b VFT, green – GABAB2 VFT, gray – illustration of the approximate position of the orthosteric binding site (PDB ID:

4MS4) (Geng et al., 2013).

The linker region between the VFT and 7TM in both of GABAB-R subunits is composed of approximately 40 residues and is not cysteine rich as in other class C GPCRs (Margeta-Mitrovic et al., 2001). The linker has not been shown to be critical for the activation and signal transduction from the VFT to the 7TM domain (Margeta-Mitrovic et al., 2001; Rondard et al., 2011). However, the distance between the C-terminus of the two LB2 subunits decreases from 45 Å to 32 Å upon activation and is thereby likely to contribute to changes in the orientation of the two 7TM domains relative to each other (Geng et al., 2013; Lecat-Guillet et al., 2017). The transmembrane part of GABAB2 hosts an allosteric binding site as shown by binding studies of the isolated GABAB2 subunits (Binet et al., 2004). It is also well demonstrated by studies manipulating the receptor composition that the GABAB2 subunit is responsible for G-protein binding (Galvez, 2001).

1.4.2 GABAB1 orthosteric binding site and ligand recognition

Agonists bind in a pocket located in the crevice of LB1 and LB2 of GABAB1a/b (Fig. 6 and Fig.

9), and induce large conformational change into the GABAB1a/b VFT such that the LB1 and LB2 interact and form a stable closed conformation in timescales necessary for full receptor activation (Geng et al., 2013; Møller et al., 2017).

Residues located in LB1 are responsible for anchoring both agonists and antagonists in the binding pocket (Geng et al., 2013). The LB1 residues Trp65, Ser130, Gly151, Ser153, His170, and Glu349 interact with both agonists and antagonists (Fig. 9). Mutational studies followed by radioligand - and [³⁵S]GTPgS - binding assays showed that the mutation of Trp65 to Ala abolished the effects of ligand binding and function of the receptor (Geng et al., 2013). Mutating His170 to Ala prevented antagonist binding, but had less effect on agonist binding (Geng et al., 2013).

Interactions with Tyr250 in LB2 seem to be unique for agonists (Evenseth et al., 2019; Geng et al., 2013), while Trp278 located in the same domain interacts with high affinity but not low affinity antagonists, in addition to agonists (Fig. 9) (Froestl, 2010; Geng et al., 2013).

Interactions with residues both in LB1 and LB2 are likely to be a requirement for agonist activation, and causes the agonists to become buried within the closed receptor. This is supported by mutational studies showing that Trp278 and Tyr250 were critical for agonist binding with less effect on binding of antagonists (Galvez et al., 2000; Geng et al., 2013).