GABA(A)R-beta3 Homopentamer (PDB ID: 4COF) from Homo sapiens
Created by: Julia Kuno
The GABAAR-β3 homopentamer (PDB ID: 4COF) from Homo sapiens is a transport protein that affects neuron signaling in the human brain by binding to the neurotransmitter GABA. Once a complex is formed, these type-A γ-aminobutyric acid receptors (GABAARs) open their anion-selective chloride channels to create temporary hyperpolarization across the membrane, thereby producing an inhibitory response in a cell (1,2). The exact mechanism describing how the neurotransmitter causes the opening of the chloride channels is not yet known, but it is hypothesized to involve a large conformational change in the protein (2). Abnormal behavior in GABAARs can lead to hyperactive neurological disorders such as anxiety and epilepsy (1). GABAARs belongs to the superfamily of pentameric ligand-gated ion channels (pGLIC), the members of which all convert chemical signals to electrical signals and share a similar pattern in their secondary structures of having 13 residues situated between two Cys residues, creating a Cys loop (2,3). Due to the fast-acting nature of pGLICs and their important roles within the nervous system, they are often targets for drug complexes (3). GABAAR in particular is known to be a target for propofol and benzodiazepine, among other drugs that regulate its activity (1,2). Propofol and benzodiazepine both heighten activity of the GABAAR by increasing the likelihood of GABA binding, thus acting as positive allosteric modulators (2,4).
The GABAAR-β3 homopentamer was obtained as a wild type protein through a series of reactions. It was first necessary to cleave the intracellular loop binding transmembrane helices 3 and 4, which was then followed by substitution of Gly-308 to Asn-421 to produce a species able to be crystallized, known as GABAAR-β3cryst (1). Crystallization was accomplished through the use of detergent, and fortunately for analysis purposes, many features of the original species were maintained throughout this process, such as the ability to form a complex with GABA (1). Complete crystallization was not possible however, as the fragments between residues 26-312 and 447-473 were lacking in the final product (1). Ultimately, structural data was obtained through x-ray diffraction, analyzed at 3-Å resolution (1).
The modulating function of the GABAAR-β3 homopentameter is directly related to its structure. According to Expasy, the GABAAR-β3 homopentameter has a molecular weight of 40795.16 Da and an isoelectric point of 6.52 (5). As indicated by its classification as a homopentameter, GABAAR-β3 contains five identical subunits, and these subunits are arranged in a circle around a central pore (2). In nature, heteromeric receptors are more prevalent, and these are hypothesized to most often consist of two α, two β, and one γ subunit (1,2). Half of each subunit, including the portion that contains the Cys loop characteristic of the pLGIC family and the N-terminus, is exposed to the surface of the cell (2). The other half is composed of four transmembrane sequences, labeled M1-M4 (2). The binding of GABA occurs in the extracellular space between subunits, resulting in a total of five neurotransmitter binding sites per protein (1). This interaction instigates conformational changes that subsequently open ion channels (1). Analysis of the β3 subunit, of which the protein here is completely composed, allowed for the establishment of the GABAAR’s ion selectivity (1). The secondary structure of the GABAAR-β3 homopentameter contains a variety of beta sheets, alpha helices, and random coils, all of which are stabilized by hydrogen bonds both between specific residues and between ligand and substrate. Hydrogen bonding between residues is evident, as shown between residues Asp-95, Ser-156, and Tyr-157; Glu-190 and Arg-192; and Arg-269 and Asp-282 (1). Notable hydrogen bonds that stabilize the ligand occur between the amidinium group of benzamidine and Glu-155, Ser-156, and Tyr-157 (1). This ligand, benzamidine, was originally added in the crystallization process simply in the attempt to form high-resolution crystals (1). However, during this process it was discovered that benzmidine acts as an agonist and binds to the receptor, inducing desensitization by closing the ion channel (1). This revelation was noteworthy as benzamidine was previously only considered to function as an inhibitor (1). Thus, the possibility of manipulation for the synthesis of other modular proteins and drugs therapies was created based on this novel finding (1). Three other ligands are associated with the GABAAR-β3 homopentamer: beta-D-mannose, N-acetyl-D-glucosamine, and the chloride ion. Neither beta-D-mannose nor N-acetyl-D-glucosamine contributes to biological function, as they were attached to glycosylation sites following endoglycosidase F1 treatment (1). The chloride ion was added to verify anion-binding sites at subunit interfaces, and it is hypothesized to function as a stabilizer of general pGLIC assembly, based off of the similarly structured bacterial GLIC (1).
As for the amino acid sequence of the GABAAR-β3 homopentamer, there are several residues that have been identified as contributing directly to the protein’s function of binding the neurotransmitter GABA. In its closed and inactivated conformation, Arg-207 serves to stabilize the GABAAR-β3 homopentamer through the formation of salt-bridges (1). Residues Glu-153 and Glu-155 act in an identical fashion (1).The segment of residues from Ala-248 to Thr-263 forms a funnel of the transmembrane M2 helix, which surrounds the ion channel (1,2). This sequence determines whether the GABAAR-β3 homopentamer is in a desensitized state, based on its width and ability for chloride anions to pass through (1). The functioning of His-267 is two-fold, as it can simultaneously coordinate with the activator propofol or Zn2+ cation inhibitors, as well as form a salt-bridge with Glu-270 that stabilizes the extracellular portion of the ion channel (1). Asn-149 serves as an N-linked glycosylation site that is found in every subunit, substitution of which lowers binding capability with GABA (1). A change in conformation of Tyr-299, which is located in the intracellular region of the ion channel, can lead to channel desensitization (1). It is also a hypothesized drug target. Additionally, there are residues that are known to aid in widespread assembly stabilization and specificity. For example, the salt-bridge extending from Arg-26 and Asp-17 to Asp-24 and Lys-13 holds together β-β subunits (1).
Two databases were utilized to compare the structures of GABAAR-β3 homopentamer to other proteins: Position-Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) and the Dali Server. PSI-BLAST finds proteins with similar sequences to the one being searched by comparing different proteins’ primary structures with the query protein and identifying gaps where the two differ in amino acid selection. From this data, E values are assigned based on how much the two sequences are alike. Significance is determined by an E value of less than 0.05, and an E value of 0.0 indicates that the two proteins are identical (6). The Dali server also analyzes similarities between proteins, but focuses on tertiary structure instead of primary. This database utilizes a sum-of-pairs method in order to discern similarity in intramolecular distances of different proteins. For this database, a Dali-Z score above 2 is significant (7). Based off of searches using both of these resources, alpha-1 glycine receptor (PDB ID: 3JAD) from Danio rerio (zebra fish) was isolated as a comparison protein due to its low E value of 3e-117 and high Z score of 33.0 (6,7). This protein belongs to the same superfamily as GABAAR, the pGLIC family, and therefore shares many features such as its five-subunit structure and classification as an alpha-helical membrane protein. However, the principal difference between the two is that the alpha-1 glycine receptor binds to the neurotransmitter glycine, whereas GABAAR is a transport protein for the neurotransmitter GABA. This is primarily due to the alpha-1 glycine receptor exploiting the N-terminal domain of its α subunit in ligand binding and the GABAAR binding its neurotransmitter in the extracellular space between subunits (8). This difference in functionality highlights the principle that structure alone cannot be used to identify protein behavior.
There are many abnormalities that
can occur with the GABAAR-β3 homopentamer that have significantly
detrimental health effects to humans. An example is the N85D mutation, which
manipulates the β5-β5’ loop and has been associated with epileptic
encephelathopathies (1). The mutations of D95N and E155G have also been linked
to this disease (1). For many other mutations, the specific conformational changes
are not known. A mutation at the residue Arg-192 has been correlated with
chronic insomnia, but the precise mechanism by which it acts has not been
identified (1). F221W has been shown to inhibit the anesthetic potentiation
effects of propofol (4). Both missense mutations such as R43Q and K289M, as
well as nonsense mutations like the premature termination of a sequence at
Gln-351, are linked to idiopathic epileptic syndromes (9). Knowledge of the
specific points of mutation that can cause certain diseases such as these have
aided remarkably in the advancement of drug synthesis for the GABAARs.