Gs-alpha
Created by Evelywn Jones
Gs-alpha (PDB ID: 1azt) is a stimulatory enzyme that belongs to a family of proteins known as heterotrimeric G proteins which transduce extracellular signals by interacting with specific intracellular effectors (2). Isolated from Bos taurus, Gs-alpha becomes active upon binding of the guanine nucleotide, guanosine-5’-triphosphate (GTP), and becomes inactive by hydrolysis of GTP to guanosine -5’-diphospahte (GDP) (9). In its active form, Gs-alpha binds to β-adrenergic receptors which are localized on plasma membranes in order to stimulate production of the enzyme adenylyl cyclase (6). As an effector, adenylyl cyclase propagates production of cyclic 3’-5’-adenosine-monophosphate (cAMP), a second messenger that plays a critical role in many physiological processes such as the rate of heart contraction, memory, and proper functioning of the endocrine system (9). Gs-alpha has a molecular weight of 93273.07 Da and an isoelectric point (pI) of 5.86.
Since all G proteins are heterotrimeric, they each contain three subunits: α,β, and γ (6). The α subunit is unique to each G protein and contains the catalytic site for recognition of specific effectors (9). In Gs-alpha, the alpha subunit is specialized in sequence and conformation for the recognition of adenylyl cyclase (9). In order to for the activation of adenylyl cyclase to be initiated, the α subunit must disassociate from the β and γ subunits (6). When Gs-alpha is bound to GDP in its inactive form, the α subunit is directly linked to the β and γ subunits (collectively known as the βγ complex) (2). As Gs-alpha becomes bound to its G protein coupled receptor, β-adrenergic receptor, its GDP is exchanged for GTP (6). Actively bound to GTP, Gs-alpha will dissociate from βγ, exposing the regions on the alpha subunit that are critical for adenylyl cyclase binding and activation (2).
Gs-alpha contains three different ligands that assist the protein in its catalytic function. Within the nucleotide-binding pocket, magnesium ions and GTPγS ligands are bound (1). Magnesium ions are often found coordinated with water, and one ion is thought to interact with one of the phosphoryls of GDP (3). This interaction involves a salt bridge that links Asp-223 to magnesium which is attached to GDP, holding Gs-alpha in its inactive form (3). Magnesium is also found localized to residues 54 and 204 (11). The GTPγS ligand is a non-hydrolyzable analog of GTP. In order to determine the crystal structure of Gs-alpha, the protein was complexed with GTPγS (1). The analog allowed Gs-alpha structure to be determined while in the active state, when the α subunit had disassociated from βγ and was capable of binding adenylyl cyclase (1). GTPγS binds to residue segments 47-54, 198-204, 223-227, and 292-295 (11). In residues 292-295, the consensus sequence of Asn-Lys-Lys-Asp (292-295) is essential to binding the guanine nucleotide (3). The third type of ligand bound to Gs-alpha is phosphate (1). The 16 phosphate anions help to stabilize the interface of the homodimer conformation of the α subunit. Gs-alpha adopts the homodimer conformation because it contains two chains that are exactly alike. The two chains are further stabilized by the localization of two of the sixteen phosphate anions at the N-terminal ends of the two chains (1).
The secondary of structure of Gs-alpha contains a large amount of helical portions. Specifically, 41% of Gs-alpha is helical with 168 residues forming 21 helices. Ten percent of Gs-alpha is also composed of β-sheets with 41 residues forming eight strands. These numbers pertain to only one chain of the α subunit. In any case, the numbers also show that over half of the protein (over half of the 402 residues that makeup a chain in the α subunit) exists as either an α helix or β sheet conformation. The rest of the residues that are not involved in α helix or β sheet structures, are instead found as random coils interspersed between the α helices and β sheets.
The overall structure of Gs-alpha can be broken down into three domains. First, there is the ras-like GTPase domain which is responsible for the hydrolysis of GTP to GDP in order to inactivate Gs-alpha. Second, there is the α helical domain which contains a region of the protein solely dominated by α helices. Third, there is the N-terminal domain, which is α helical and extends away from the rest of the protein. Within these domains are three switch elements (I, II, and III) that are key to activation of adenylyl cyclase. Regions common to all G proteins for effector interaction are the switch regions, the αN-β1 strand, and the α3 helix which are all on the surface of the protein facing the direction of the βγ subunits; therefore, these regions are highly conserved among the G proteins. In Gs-alpha, adenylyl cyclase activity depends on the C-terminus of the α2-helix located in the switch II region, the C-terminus of the α3-β5 loop in the switch region, and the α4-β6 loop. These regions all face upwards toward the cytoplasmic face of the plasma membrane because that is where Gs-alpha comes into contact with the β-adrenergic receptor (1). Reference Figure 1 in order to further visualize the locations of these domains, switch elements, and loops.
Specificity for adenylyl cyclase depends on the primary structure of the α-helices and β sheets that compose Gs-alpha. The α3-β5 loop is unique to Gs-alpha in the fact that this loop is rotated downwards to allow formation of a hydrophobic pocket on the back of the β sheet that is filled by Met-386 of the α5 helix. The rotation is supported by movement of Leu-282 towards a conserved Phe-238. Both the α3-β5 and α4-β6 loops are supported by a stacking interaction of Trp-277 and His-357. These few, specific residues are critical for separating Gs-alpha from another G-protein (1).
The conformation of the three switch elements in Gs-alpha are dependent on which guanine nucleotide, GTP or GDP, is bound to the protein. The guanine nucleotide itself is bound in the nucleotide binding pocket located between switch I and switch II. It is in this pocket that magnesium ion is also found coordinated not only by water, but also by the side chains of the residues composing the pocket. One such residue in the binding pocket is Arg-201, which serves to stabilize the phosphate intermediate during GTP hydrolysis (1). Other amino acids that are essential to binding of adenylyl cyclase are Gln-237 and Gly-49. Mutations inGln-237 show that adenylyl cyclase is constitutively activated because the GTP form of Gs-alpha could not be hydrolyzed (5). Mutations in Gly-49 show the opposite effect, in that not much adenylyl cyclase is stimulated at all, suggesting an impairment in binding of GTP (5).
The residues that compose the adenylyl cyclase activation site are highly conserved in the α2 helix and α3-β4 loops of the switch II element. Of the nine residues that are essential to binding adenylyl cyclase, seven are constant for all Gα proteins. However, two residues, His-236 and Glu-239, differ from the rest of the Gα proteins, so that only Gs-alpha shows true adenylyl cyclase activation. Switch III which is associated with switch II by ionic interaction does not play any role in adenylyl cyclase binding. Gs-alpha does not undergo a conformational change upon binding adenylyl cyclase because the bound GTP or GTPγS resists any change in the active form of the protein. The switch II element does, however, undergo a significant conformation change when GDP is exchanged for GTPγS. In the molecule of Gs-alpha, the change is seen as a disassociation of the α subunit from the βγ complex. From this fact, it is predictable when GTP bound Gs-alpha is hydrolyzed, it will convert back to the GDP conformation in which the same site that bound adenylyl cyclase will then bound the βγ complex. Some residues which are essential for βγ binding are Ile-184, Phe-199, Lys-210, Trp-211, Cys-214, and Phe-215. The difference between the effector and βγ binding sites is that the effector will bind to the same residues as the βγ does, but it will also form contacts with the α3-β5 and α4-β6 loops of which the βγ complex does not interact (1).
In Gs-alpha, residues 236-356 compose the shortest linear chain that is required for activation of adenylyl cyclase. Of those amino acids, only those located in the N-terminus half (236-296) are essential for initial activation of adenylyl cyclase. The residues involved in binding the effector are located on the membrane-facing side of the molecule (towards the cytoplasm) where GDP is also exchanged for GTP. Within this linear chain, the residues responsible for activation of adenylyl cyclase are not continuous, but grouped into four different regions. Region one included residues Gln-236, Asn-239, Glu-240, region two included Asn-261 and Gln-262, region three included Trp-277, Arg-280, Leu-282, Thr-284, Ile-285, and region four included Ser-349 to Arg-356. Mutations in these regions lead a loss of Gs-alpha function, so that Gs-alpha will either not bind adenylyl cyclase or prevent adenylyl cyclase from producing cAMP (2).
Gs-alpha contains a palmitate fatty acid thioester-linked to its N-terminus. Palmitate is a 16 carbon fatty acid that is covalently attached to an N-terminal residue of Cys-3. Ordinarily, palmitoylation is observed in membrane attachment. Therefore, the palmitoylation of Gs-alpha is necessary in order to link it to the G-protein coupled receptor, β-adrenergic receptor which is located in the plasma membrane. Although palmitoylation links Gs-alpha to the membrane it is not a requirement for activation of adenylyl cyclase. In fact, Gs-alpha proteins that lack a palmitate at their Cys-3 ends only show reduced activation of adenylyl cyclase and production of cAMP, but nonetheless, still produce some cAMP (4).
Single residue mutations in Gs-alpha are enough to cause severe, life-threatening illness and disease in humans. One of these mutations involves Arg-201 which serves as a key residue in the GTPase domain of Gs-alpha. Arg-201 can be mutated by cholera toxin in which the residue becomes adenosine diphosphate ribosylated (ADP). The attachment of ADP to Arg-201 will cause an inhibition in the GTPase activity of Gs-alpha. This means that Gs-alpha will be unable to hydrolyze GTP and will constitutively activate adenylyl cylase, which in turn overproduces cAMP. The result on the body is that epithelial cells within the intestines become dehydrated and cause diarrhea. This single alteration of Arg-201 produces the deadly disease known as cholera (1).
Single mutations in Gln-227 and Arg-201contribute to the growth of tumors in the pituitary and thyroid glands causing the McCune-Albright syndrome (1). In this case, mutation of Arg-201 and Gln-227 occurs at the nucleic acid sequence that encodes those residues in Gs-alpha (10). In the nucleic acid sequence, a single base pair is replaced, resulting in a histidine instead of arginine at position 201 and an arginine instead of a glutamine at position 227 (10). The results of these substitutions are that GTP cannot be hydrolyzed and adenylyl cyclase is constitutively activated (10). This overproduction in cAMP can cause an over stimulation in hormone production by the endocrine glands. Since the cancer-producing-defect in Gs-alpha is initially caused by a nucleotide sequence substitution, the mutated Gs-alpha is called an oncogene (10).
Another protein that has a high sequence homology with Gs-alpha is the enzyme, guanine nucleotide-binding protein G (i), alpha-1 subunit (PDB ID:2zjy). Results from BLAST (E=0.0) and DALI (Z=39.0) indicate that the primary and tertiary structures of this protein are very similar to Gs-alpha (8). Gi-alpha, from Rattus norvegicus, is part of the same G protein family as Gs-alpha; therefore, Gi-alpha is architecturally very similar to Gs-alpha. Unlike Gs-alpha which stimulates adenylyl cyclase activity, Gi-alpha serves to inhibit adenylyl cyclase. In terms of secondary structure, Gi-alpha is 44% helical with 18 helices from 159 residues and 13% β sheets with 8 strands from 49 residues. Gi-alpha is attached to tetrafluoraluminate (AlF4-), GDP, and magnesium ion.
Gi-alpha proteins differ significantly from Gs-alpha by the fact they are stimulated by the regulators of G protein signaling (RGS) family of proteins. Specifically, RGS proteins alter the GTPase activity of Gi-alpha but have no effect on Gs-alpha. In Gi-alpha the α3-β5 loop is rotated upward, the α4-β6 loop is shorter and contains a different sequence, and the α5-helix is straight in comparison to the same structural features in Gs-alpha. In the catalytic domain responsible for binding adenylyl cyclase, two residues vary from that of Gs-alpha: Gln-236 and Asn-239. The reason that Gi-alpha does not bind adenylyl cyclase is because its α3-β5 loop and switch II element are positioned away from each other, so that adenylyl cyclase cannot bind to both sites. The displacement arises because α3-β5 loop of Gi-alpha contains a bulky Phe instead of Leu-282(1).
The difference between Gs-alpha and Gi-alpha in effector recognition for adenylyl cyclase may be due to the amino acid sequence in the RGS binding domain of both molecules (1). In Gi-alpha, RGS proteins activate GTP hydrolysis by binding to and stabilizing all three switch elements of Gi-alpha while in its transition state conformation (7). RGS molecules serve to accelerate the GTPase activity of Gi-alpha; however, this effect is not seen with RGS proteins that bind to Gs-alpha (1). RGS proteins specify what kind of G proteins to bind to by recognition of the side chains located on the switch elements (7). For example, in Gi-alpha there are six residues that come into contact with RGS4: Lys-180, Thr-182, Val-185 in switch I and Ser-206, Lys-209, His-213 in switch II (1). All of these positions in Gs-alpha are substituted so that steric overlap, charge repulsions, and concavity in the chain defer RGS proteins from successful binding to Gs-alpha (1).
Guanine nucleotide-binding protein G(k) subunit alpha (PDB ID: 2ode) is another enzyme with significant sequence similarity to Gs-alpha. The results from BLAST (E=0.0) and DALI (Z= 39.3) show that Gk-alpha has an almost identical primary structure and a closely related tertiary structure to Gs-alpha (8). Gk-alpha is an enzyme that is found in Homo sapiens and contains the same attached ligands as Gi-alpha: AlF4-, magnesium ion, and GDP. Thesecondary structure of Gk-alpha is 46% helical with 161 residues making up 18 helices and 13% beta sheet with 48 residues composing 8 beta strands. Just like with Gi-alpha, the secondary structure matches very closely with Gs-alpha. In humans, Gk-alpha plays a very big role in regulating RGS8 which acts to accelerate GTP hydrolysis (7). In the body, this particular RGS protein plays a part in regulating the production of hormones, vision, neurotransmission, and olfaction (7).
As part of a large G-protein family, Gs-alpha shares a large portion of tertiary structure similarity with quite a few other G-proteins. Once such protein is known as Type V adenylyl cyclase (PDB ID: 1cul) which also contains a guanine nucleotide-binding protein Gs. Type V adenylyl cyclase contains two inhibition sites, in which case its functions are similar to Gi-alpha. In fact, there is evidence that switch II of Gi-alpha and its α4-β6 loop are involved in formation of type V adenylyl cyclase (1).The secondary structure of type V adenylyl cyclase is 35% helical (7 helices, 76 residues) and 25% beta sheet (9 strands and 55 residues). According to DALI (Z=48.7), the tertiary structure of this enzyme is quite similar to Gs-alpha (8).
Transducin-alpha (PDB ID: 1tad) is another guanine nucleotide protein that has a common tertiary structure to Gs-alpha as indicated by Dali (Z=39.6) (8). The secondary structure of transducing-alpha49% helical and 12% beta sheet in which 18 helices are composed from 161 residues and 8 strands are composed from 42 residues.
G protein coupled receptor kinase 2 (PDB ID: 2bcj)is an enzyme that serves to phosphorylate other GPCRs so as to desensitize them to an extracellular signal and terminate signal transduction. Within this protein is a guanine nucleotide binding protein G (q) alpha subunit, which activates the enzyme phospholipase C. Results from DALI (Z=39.4) show that this Gq-alpha proteins shares a very similar tertiary structure with Gs-alpha (8). The secondary structure of Gq-alpha is 40% helical and 13% beta sheet. There are 279 residues that make up the 28 helices and 93 residues that makeup the 19 strands of Gq-alpha.
Although G-proteins share a significant amount of their structure, specific residue variants among the proteins specify recognition by different effectors as shown by Gs-alpha. The uniqueness of these G-proteins allows for cells to accomplish the process of signal transduction efficiently and directly.