Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates
Introduction
The Cys-loop superfamily of ligand-gated ion channels (LGICs) includes nicotinic acetylcholine (nAChR), GABAAR, glycine (GlyR), and 5-hydroxytryptamine (5HT3) receptors [1], [2], [3], [4], [5], [6]. Most studies of these receptors, using site-directed mutagenesis and chemical labeling techniques, have focused on either the ligand-binding domain (LBD) or the transmembrane domain (TMD). More recently, however, it has become clear that a description of the interaction between these domains will be necessary to understand how agonists activate the ion channels [7] as well as how anesthetics and alcohols may alter this process [1], [8], [9]. Fortunately, preparation of a comparative model that combines both domains has been made possible by the recent publication of high-resolution crystal structures of proteins that can serve as templates for both domains. Here we describe building a model of the TMD by using the structure of a pentameric ion channel to align five copies of a four-alpha helical bundle. We then merged the TMD model with our previously published model [10] of the LBD that was built by threading the sequence of GABAAR onto the crystal structure of an acetylcholine binding protein (AChBP) from the snail Lymnaea stagnalis [11]. Despite being derived using a completely different technique, much of our model agrees with the overall form of the recently published model of the homologous torpedo nAChR that was derived by aligning four polyalanine alpha helices onto the electron density of a 4 A resolution cryoelectron micrograph [12]. The additional value of our model comes from illustration of features not visible in the nAChR model at its present moderate resolution, especially the loops that connect alpha helices in the TMD with loops in the LBD. In addition, our model successfully incorporates and explains a large body of information derived from site-directed mutagenesis, photoaffinity labeling, as well as anesthetic and alcohol binding. Our model of GABAAR will allow comparison with the overall morphology of nAChR deduced by cryoelectron microscopy. In particular, it will be possible to compare modes of anion-conductance in GABAAR and GlyR with cation-conductance in nAChR. This model will also serve as a platform from which future site-directed mutagenesis studies can be planned and theories of anesthetic action tested.
Section snippets
Methods
We built our model in six steps: (1) a four-alpha helical bundle found in the crystal structure of bovine cytochrome c oxidase (2OCC) [13] was identified as a template for the TMD of a single subunit. (2) The five pore-forming alpha helices of a bacterial mechanosensitive channel (1MSL) were used as a template for the pentameric ion channel of GABAAR (PDB entry 1MSL) [14]. (3) Five copies of the tetrameric subunit were packed according to a partial alignment with the pentameric template to
Geometry and dimensions
The overall dimensions of the combined model (Fig. 2A and B) approximate the electron density envelope in the cryoelectron micrographs of nAChR [12], [21], [22]. In addition, there was a smooth transition between the end of the ion pore of the TMD and the beginning of the pore in the LBD (Fig. 2B). The C-terminal residue of the LBD made an amide bond with the N-terminal of the TMD with very little stretching or distortion of the structure (Fig. 2B). Globally, it appears that the comparative
Discussion
Our model of a GABAAR was constructed using homology modeling based on the three templates. It was further refined using a variety of experimental results as initial constraints. However, even with restraints removed our combined model continued to satisfy a great deal of experimental data. This includes the alpha-helical characteristics of the TMD (Fig. 1B) and the appropriate orientation of residues for specific types of labeling experiments. There was excellent fit of the LBD with the TMD (
Conclusions
A model of the GABAARa1 built from molluscan, bacterial, and mammalian templates satisfies most experimental criteria for shape, alpha-helical content, exposure of specific residues in labeling experiments, the location of the ion selectivity filter, possible sites of anesthetic and alcohol binding, and the transduction of agonist binding from the LBD to the TMD. This model is also among the first to present atomic level details of an entire LGIC complex containing both LBD and TMD. In the
Acknowledgement
The research was funded by NIH grants RO1 GM63034 and RO1 AA013378 to JRT, and by the Veterans Administration Health Care System to EB. We wish to thank Dr. Douglas Brutlag for reading the manuscript and Drs. Wayne L. Hubbell, Barry Honig, R. Adron Harris, and Neil Harrison for helpful discussions.
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Present address: Palo Alto Veterans Affairs Health Care System, Palo Alto, CA 94304, USA.