Ligand Binding Mode of GABA A Receptor- Associated Protein Oliver H. Weiergräber 1 , Thomas Stangler 1,2 , Yvonne Thielmann 1 , Jeannine Mohrlüder 1,2 , Katja Wiesehan 1 and Dieter Willbold 1,2 ⁎ 1 Institut für Neurowissenschaften und Biophysik, Molekulare Biophysik, Forschungszentrum Jülich, D-52425 Jülich, Germany 2 Institut für Physikalische Biologie und BMFZ, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany Received 15 May 2008; received in revised form 27 June 2008; accepted 30 June 2008 Available online 7 July 2008 The γ-aminobutyric acid type A (GABA A ) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA A receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 Å resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA A receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein. © 2008 Elsevier Ltd. All rights reserved. Edited by K. Morikawa Keywords: GABARAP; GABA A receptor; synthetic peptide; phage display; X-ray crystallography Introduction The neurotransmitter γ-aminobutyric acid (GABA) is one of the principal mediators of hyperpolarizing synaptic activity in the mammalian central nervous system. 1 It exerts its function via three major types of cell-surface receptors: while GABA A and GABA C receptors are ligand-gated ion channels with chloride conductance, GABA B receptors are G-protein- coupled receptors modulating the activity of potas- sium channels. 2 The GABA A receptor-associated protein (GABARAP) has been identified in a two- hybrid search for proteins interacting with the long cytoplasmic loop connecting transmembrane do- mains (TM) 3 and 4 of the GABA A receptor γ2 sub- unit. 3 The significance of this association was supported by coimmunoprecipitation as well as in vitro binding assays. Interestingly, coexpression of GABARAP with the GABA A receptor in quail fibroblasts led to increased clustering of receptors at the cell surface as well as altered channel kinetics, 4 suggesting a role for GABARAP in the organization of GABAergic synapses. In contrast, immunostaining and electron microscopy with cultured neurons revealed that GABARAP is not enriched at inhibitory synapses to a significant extent. 5 Instead, the protein is predominantly found in a perinuclear region corresponding to endoplasmic reticulum and Golgi membranes, with lower levels in tubulovesicular structures beneath the postsynaptic membrane. In the same study, GABARAP was shown to colocalize with N-ethylmaleimide-sensitive factor, which plays an important role in vesicle transport and fusion events. 5 Moreover, overexpression of GABARAP in neurons was demonstrated to increase the amount of GABA A receptors detected at the cell surface. 6 These *Corresponding author. Institut für Physikalische Biologie und BMFZ, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany. E-mail address: d.willbold@fz-juelich.de. Abbreviations used: ESRF, European Synchrotron Radiation Facility; GABA, γ-aminobutyric acid; GABARAP, GABA A receptor-associated protein; GATE- 16, Golgi-associated ATPase enhancer of 16 kDa; GST, glutathione S-transferase; HSQC, heteronuclear single quantum coherence; LC3, light chain 3 of microtubule- associated protein 1; NCS, noncrystallographic symmetry; PDB, Protein Data Bank; PE, phosphatidylethanolamine; SPR, surface plasmon resonance; TM, transmembrane domain. doi:10.1016/j.jmb.2008.06.086 J. Mol. Biol. (2008) 381, 1320–1331 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.