Structural basis for auxiliary subunit KCTD16 regulation of the GABA _B receptor

Abstract

Metabotropic GABA B receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABA B receptor complexes contain the principal subunits GABA B1 and GABA B2 , which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABA B receptors and modify the kinetics of GABA B receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABA B receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABA B2 receptor. A single GABA B2 Cterminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABA B2 -KCTD16 interface disrupted both the biochemical association and functional modulation of GABA B receptors and G protein-activated inwardly rectifying K + channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABA B receptors. Defining the binding interface of GABA B receptor and KCTD reveals a potential regulatory site for modulating GABA B -receptor function in the brain. GABA B receptor | KCTD | principal and auxiliary subunits | crystal structure M etabotropic γ-aminobutyric acid (GABA) type B (GABA B )receptors are implicated in various neurological and psychiatric disorders, including spasticity, epilepsy, depression, addiction, and anxiety (1-3). GABA B receptors provide a crucial component of inhibitory neurotransmission in the nervous system (1-3), via coupling to G i/o type G proteins that modulate three different downstream effectors: voltage-gated Ca 2+ channels, G proteinactivated inwardly rectifying K + (GIRK) channels, and adenylyl cyclase (1-3). The GABA B receptor functions as an obligatory heterodimer, consisting of the GABA B1 and GABA B2 subunits (4-9), whereby the GABA B1 subunit binds orthosteric ligand (10, 11) and the GABA B2 subunit couples to the G protein (12)(13)(14)(15)(16)(17)(18).Recent proteomic studies led to the discovery of a family of auxiliary proteins for the GABA B receptor, originally referred to as potassium channel tetramerization domain-containing (KCTD) proteins (19)(20)(21). A subset of KCTD proteins (numbered 8, 12, 12b, and 16) interact with the cytoplasmic tail of the GABA B2 subunit (3, 20, 21) and convey unique functional properties to the signaling of the GABA B receptor (3). For example, expression of each of the four KCTD proteins leads to acceleration of GABA B receptordependent activation of GIRK currents, albeit to different extents (20,22). KCTD12 and KCTD12b also promote rapid desensitization of the GABA B receptor-induced GIRK current, by uncoupling Gβγ from GIRK channels (20,22,23). By contrast, KCTD8 and KCTD16 generate primarily nondesensitizing receptor responses (20,22...

The organizing principle of GABAB receptor complexes: Physiological and pharmacological implications

“…Of note, charged interactions at the pentameric interface of the KCTD16 T1 structure are conserved among all GABA B ‐related KCTD proteins, which explains how pentameric heteromers can form. Moreover, conservation of amino acids in the GB2/KCTD interface is compatible with the observation that KCTDs can form heteromers that regulate GBR responses . In fact, the formation of KCTD heteromers enables a fine‐tuning of receptor kinetics.…”

“…The cytosolic K + channel tetramerization domain (KCTD) proteins KCTD8, KCTD12, KCTD12b and KCTD16 bind to the C‐terminal domain of GB2 (Figure ). Recent cryo‐electron microscopy and crystallization studies indicate that the T1 tetramerization domain of KCTD16 assembles into an open pentameric ring with an inner diameter of ~25 Å . Adjacent KCTD16 T1 subunits are arranged side‐by‐side with similar C‐ and N‐terminal orientation.…”

“…Adjacent KCTD16 T1 subunits are arranged side‐by‐side with similar C‐ and N‐terminal orientation. The high‐resolution crystal structure of the T1 pentamer in complex with a C‐terminal peptide of GB2 shows that a single GB2 peptide binds to the inner surface of the open pentameric ring, with the interface having exceptionally high shape complementarity . The GB2 peptide is located asymmetrically, off centre, away from the opening of the ring .…”

“…The high‐resolution crystal structure of the T1 pentamer in complex with a C‐terminal peptide of GB2 shows that a single GB2 peptide binds to the inner surface of the open pentameric ring, with the interface having exceptionally high shape complementarity . The GB2 peptide is located asymmetrically, off centre, away from the opening of the ring . The GB2 peptide loops around inside the ring structure of the pentamer with the N‐ and C‐termini of GB2 pointing to the KCTD16 N‐terminus .…”

The Structural Versatility of the BTB Domains of KCTD Proteins and Their Recognition of the GABAB Receptor

“…Indeed, the data were limited to the KCTD5 member whose pentameric structure was determined a decade ago [23]. Very recently, other structural information derived through the application of different approaches became available [19,21,24,25,26,27,28]. These studies have provided some interesting clues on the structure and partnerships of these proteins.…”