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From Proteopedia
| This Sandbox is Reserved from January 19, 2016, through August 31, 2016 for use for Proteopedia Team Projects by the class Chemistry 423 Biochemistry for Chemists taught by Lynmarie K Thompson at University of Massachusetts Amherst, USA. This reservation includes Sandbox Reserved 425 through Sandbox Reserved 439. |
IntroductionGlycogen synthase kinase-3, or GSK-3, is one of the main proteins that controls the activation of glycogen synthase. GSK-3 is serine/threonine protein kinase which regulates the phosphorylation of serine and threonine molecules. Serine/threonine kinase is important for the regulation of cell proliferation, cell death, cell differentiation, and embryonic development[1]. GSK-3 is found in two forms, GSK-3β and GSK-3α. The two forms have different functions with GSK-3 β involved in energy metabolism, neuronal cell development, and body pattern formation, while GSK-3α has more function with WNT signaling pathways, which controls cell fate. This proteopedia page will be focused on GSK-3β. The GSK-3β is found in most mammals, all with similar structure and function. In experiments when GSK-3β was perturbed in mice, embryonic lethality during gestation was demonstrated[2]. Recent research in regards to GSK-3β includes type II diabetes, Alzheimer's Disease, inflammation, cancer, and neurological disorders such as strokes and bipolar disorder. GSK-3β has been shown to interact with various enzymes including: TGF- β1, Smad3, AKAP11, AXIN1, AXIN2, AR, CTNNB1, DNM1L, MACF1 MUC1, SMAD3, NOTCH1,NOTCH2, P53, PRKAR2A, SGK3, and TSC2. This page focuses on a GSK-3β complex with a Staurosporine inhibitor. Since ATP has a stronger affinity to binding to staurosporine than to protein kinases, the molecule acts a competitive inhibitor in regards to GSK-3β[3]. This is a sample scene created with SAT to by Group, and another to make of the complex. Overall StructureThe overall structure of GSK-3β has two phosphorylation sites that are involved in catalysis. One of these sites is Ser 9, resulting in the inactivation of GSK-3β. The second phosphorylation site is Tyr 216, located on the activation loop (green ), and is responsible for the increase in catalytic activity. GSK-3β has the characteristic two-domain kinase fold, containing a N-terminal β-strand domain (light blue, residues 25-138) and a C-terminal α-helical domain (red, residues 139-343). There is an interface between the α and β domains, at which the ATP-binding site is located, encircled by the hinge and the glycine-rich loop. The activation loop (green) runs along the surface of the substrate-binding groove. There are 39 residues in the C-terminus end that are outside the main kinase fold. These residues form a small domain that closely packs next to the α-helical domain. The β-strand domain is formed by seven β-strands that run in an antiparallel formation. Strands 2-6 form a β-barrel, through which a short α helix (yellow, residues 96-102) aligns against the β-barrel [4].
Binding InteractionsBefore GSK-3β can phosphorylate a substrate, the β and α domains of the protein must align. To ensure alignment of the domains, which can either promote or decrease catalysis, GSK-3β utilizes phosphorylated residues. As mentioned in overall structure, phosphorylation of Ser 9 inhibits the catalytic ability of GSK-3β, while phosphorylation of Tyr 216 promotes catalysis by 200 fold. When Ser 9 is phosphorylated, the N-terminus of the protein acts as a pseudo-substrate, binding to the active site and preventing any catalytic activity from occurring. The inhibitor staurosporine, prevents catalytic activity by binding slightly above the active site of GSK-3β while rotating the N-terminal domain of the protein back in order to occupy a prefered binding mode. More details on the binding of staurosporine can be read in the Additional Features section. When Tyr 216 is phosphorylated, the polar residues Arg 96, Arg 180, and Lys 205 all rearranged, due to electrostatic interactions to point towards the phosphorylated molecule, allowing for the protein to fold into its active form [4]. To influence binding of substrates, a salt bridge must form between Glu 97 and Lys 85 located in the active site. GSK-3β recognizes a sequence on the substrate containing two serine molecules separated by three residues (SXXXS). If the last serine in the sequence is phosphorylated prior to its encounter with GSK-3β, also known as primed phosphorylation, then the catalytic rate increases by 100-1000 fold. Catalytic activity of primed phosphorylated substrates increases catalytic activity by replacing the phosphate ion that aligns the Arg 96, Arg 180, and Lys 205 residues. Although not all substrates that GSK-3β phosphorylated require primed phosphorylation, substrates that do have a primed serine increase catalytic activity by utilizing electrostatic interactions to hold the protein in its active form [4]. when Tyr 216 is phosphorylated, due to the electrostatic interactions between the residues and the phosphate ion.Blue atoms - cationic side chains White atoms - amino acid backbone Additional FeaturesThere are three kinds of interactions in the GSK-3β and staurosporine complex, including: direct H-bonds, water-mediated polar interactions and hydrophobic interactions .The GSK-3β and staurosporine complex shows . There are only two direct H-bonds, and they are observed between
Besides direct H-bond, the water-mediated polar interactions are observed between the carbonyl oxygen of Gln 185 and N4 (nitrogen) of the glycosidic ring. The typical hydrogen bond (H-bond) is categorized to be between 2.2 and 4.0 Å [5]. Since many pdb files lack hydrogen atoms, a significant H-bond can be considered when donor-acceptor distance are probably 3.5 Å [5]. However, the length between between Gln 185 and Strauroporine is 4.47 Å which surpasses typical H-bond distance; therefore, it forms a water mediated polar interaction between these atoms instead of direct H-bond[3]. This is a unique interaction to the GSK-3β and staurosporine complex, since other protein kinase (e.g. CDK2, Chk1, LCK, PKA) -staurosporine complexes show direct H-bond interaction between two moieties. There is a significant number of in the GSK-3β and staurosporine complex; to be more specific, this complex buries 891 Å2 surface area[3]. The hydrophobic residues significantly interact with the fuzed carbazole moiety of saurosporine. Quiz Question 1GSK-3 beta has various inhibiters; one example is AMP-PMP. These inhibitors bind to the N-terminus of the ligand on the GSK-3 beta complex, a result of the classical binding mechanism for a protein kinase. However, in the case of staurosporine (another inhibitor), it is unable to classically bind to the N-terminus of the ligand on the GSK-3 beta complex. This is because, in a GSK-3 beta complex with staurosporine, the ligand in question has an incompatible angle at the N-terminus, thus failing to undergo classical binding[3]. What type of bonding does GSK-3 beta exhibit with staurosporine, and which of its residues form this type of bond? If needed, a green screen of the complex can be found below.
Quiz Question 2What are the locations of the active sites on each isoform? These green screens may help you. Remember to zoom and rotate each scene to better understand the structures. The first scene displays the two isoforms. The second screen shows the Amino and Carboxy chain termini with a provided color key to use when viewing. The third scene shows the locations of the α-helices and β-sheets on the protein as well as the complexed staurosporene molecule. 1) 2)
3)
See Also
CreditsIntroduction - Zachary Plourde Overall Structure - Sarah Johnson Binding Interactions - Christina Lincoln Additional Features - Bach Pham & Elvan Cevac Quiz Question 1 - Nerses Haroutunian Quiz Question 2 - Nick H-K References
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