br Extracellular domain architecture The extracellular

Extracellular domain architecture The extracellular region of GABAB receptor exists in a heterodimeric configuration regardless of interactions within the rest of the protein (Geng et al., 2012, Liu et al., 2004, Nomura et al., 2008). The extracellular domains of the GABAB1 and GABAB2 subunits are each primarily composed of a Venus flytrap (VFT) module (Pin and Bettler, 2016) (Fig. 1A). The structure earns its moniker by resembling the Venus flytrap plant. In addition, molecular cloning identified two main isoforms of the GABAB1 subunit (Kaupmann et al., 1997): GABAB1a, found predominantly in the presynaptic terminal, and GABAB1b, associated with the postsynaptic terminal (Billinton et al., 1999, Gassmann and Bettler, 2012, Kaupmann et al., 1997) (Fig. 2A). These isoforms differ by the presence of two complement control protein (CCP) modules, or sushi domains, in the N-terminus of GABAB1a, but otherwise perform the same ligand-binding function through an identical VFT region (Blein et al., 2004, Hawrot et al., 1998, Kaupmann et al., 1997). The first CCP module of GABAB1a is not compactly folded according to various biophysical measurements such as circular dichroism (CD) spectroscopy (Blein et al., 2004). In contrast, the structure of the second CCP has been solved by nuclear magnetic resonance (NMR) spectroscopy, and shows a well-ordered barrel-like architecture that is stabilized by disulfide bridges (Blein et al., 2004) (Fig. 2B). The CCP domains are attached to Temsirolimus receptor containing axonal-sorting signals to localize GABAB1a to the axon terminal (Biermann et al., 2010). The crystal structures of a heterodimeric complex of GABAB1b VFT and GABAB2 VFT have been solved by x-ray crystallography (Geng et al., 2013). Within each complex, the two subunits bind in a side-by-side manner while facing opposite directions, as if they are “dancing cheek-to-cheek” (Fig. 3). The VFT module of each GABAB receptor subunit contains two domains or lobes, LB1 and LB2, with LB1 resting atop LB2 and reaching further into the extracellular space (Fig. 1, Fig. 3). These domains are joined by three loops on a single end to form a VFT-like configuration that can oscillate between open and closed conformations. The VFT module is a shared structural feature among all class C GPCRs (Pin et al., 2003). It is also found in bacterial periplasmic binding proteins (Sack et al., 1989) and ionotropic glutamate receptors (Jin et al., 2009, Karakas et al., 2009, Kumar et al., 2009). In addition, x-ray imaging has resolved the dimeric extracellular region in various ligand-bound states: ligand-free, in complex with six different antagonists and in complex with two different agonists (Geng et al., 2013). The apo- and antagonist-bound structures have nearly identical conformations and represent the receptor in the resting state. The agonist-bound structures correspond to the active receptor state. GABAB1 and GABAB2 display different conformational dynamics consistent with their distinct functional roles. It is within the interdomain crevice of GABAB1 VFT that orthosteric ligands bind (Kaupmann et al., 1997, Malitschek et al., 1999) (Fig. 3, Fig. 4). Binding of an agonist causes the GABAB1b VFT to close 29° compared with the inactive state (Geng et al., 2013). On the other hand, GABAB2 VFT is not involved in ligand recognition, despite a sequence homology of 33% with GABAB1b VFT (Kniazeff et al., 2002). As a result, the GABAB2 VFT is perpetually vacant and its interdomain cleft remains open (Geng et al., 2012, Geng et al., 2013). Despite these differences, both subunits cooperate with each other to perform signal activation. The inactive structures indicate that GABAB1b and GABAB2 subunits attach at the N-terminal LB1 domain through noncovalent interactions (Geng et al., 2013). This interface is largely conserved in the active state, suggesting that the LB1-LB1 interaction mostly serves to facilitate heterodimer formation.