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Introduction

This is a sample scene created with SAT to by Group, and another to make of the protein.

GSK-3 is a serine/threonine protein kinase which regulates the addition of phosphate molecules onto serineand threonine amino acid residues. Serine/threonine protein kinases are responsible for phosphorylating the serine/threonine kinase receptors which play a role in the regulation of cell proliferation, programmed cell death (apoptosis), cell differentiation, and embryonic development ^[1]. GSK-3 has two isoforms, GSK-3 beta and GSK-3 alpha. GSK-3 beta is more involved in energy metabolism, neuronal cell development, and body pattern formation, while GSK-3 alpha has more function with WNT signaling pathways. GSK-3 beta is found in most mammals, all with similar structure and function. In experiments when GSK-3 beta was perturbed in mice, embryonic lethality during mid-gestation was demonstrated.

GSK-3 beta has been shown to negatively regulates TGF-beta1 and Angiotensin II-mediated cellular activity through interaction with Smad3. GSK-3 beta directly interacts with Smad3, preventing its movement into the nucleolus, which don't allow it to perform cell death. This forces Angiotensin II apoptosis in cardiac myocytes^[2]. Other enzymes that this kinase interacts with are: AKAP11, AXIN1, AXIN2, AR, CTNNB1, DNM1L, MACF1 MUC1, SMAD3[ NOTCH1,NOTCH2, P53, PRKAR2A, SGK3, and TSC2^[3]. Recent research in regards to GSK-3 includes type II diabetes , Alzheimer's Disease, inflammation, cancer, and bipolar disorder. This page demonstrates a GSK-3 complex with a Staurosporine inhibitor.

Overall Structure

The 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 (shown in 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 (purple) 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 Interactions

Before 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.^[5]

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.^[6]

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 Features

There 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

• The carbonyl oxygen of Asp 133 and N¹ (nitrogen) of staurosporine. The length of this hydrogen bond is 2.93 Å.
• The backbone nitrogen of Val 135 and O⁵ (oxygen) of staurosporine. The length of this hydrogen bond is 2.76 Å.

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 ^[7]. Since many pdb files lack hydrogen atoms, a significant H-bond can be considered when donor-acceptor distance are probably 3.5 Å ^[7]. 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^[8]. 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 Ų surface area^[8]. The hydrophobic residues significantly interact with the fuzed carbazole moiety of saurosporine.

Quiz Question 1

GSK-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^[8].

What type of bonding does GSK-3 beta exhibit with staurosporine, and which of its residues form this type of bond? A green screen of the complex as well as a lewis structure of the staurosporine molecule are found below, if needed.

Quiz Question 2

What 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.

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See Also

• UvrABC
• Glycogen Synthase Kinase 3
• Serine/Threonie Protein Kinase
• Glycogen Synthase Kinase 3 Beta complexed with Axin Peptide and Inhibitor 7d
• Glycogen Synthase Kinase 3 Beta complexed with AMP-PNP
• Glycogen Synthase Kinase 3 Beta complexed with Axin Peptide and Leucettine L4
• Glycogen Synthase Kinase 3 beta (GSK3B) complexed with a ruthenium octasporine ligand (OS1)

Credits

Introduction - 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

1. ↑ Capra M, Nuciforo PG, Confalonieri S, Quarto M, Bianchi M, Nebuloni M, Boldorini R, Pallotti F, Viale G, Gishizky ML, Draetta GF, Di Fiore PP. Frequent alterations in the expression of serine/threonine kinases in human cancers. Cancer Res. 2006 Aug 15;66(16):8147-54. PMID:16912193 doi:http://dx.doi.org/10.1158/0008-5472.CAN-05-3489
2. ↑ Bertrand JA, Thieffine S, Vulpetti A, Cristiani C, Valsasina B, Knapp S, Kalisz HM, Flocco M. Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors. J Mol Biol. 2003 Oct 17;333(2):393-407. PMID:14529625
3. ↑ Hua F, Zhou J, Liu J, Zhu C, Cui B, Lin H, Liu Y, Jin W, Yang H, Hu Z. Glycogen synthase kinase-3beta negatively regulates TGF-beta1 and Angiotensin II-mediated cellular activity through interaction with Smad3. Eur J Pharmacol. 2010 Oct 10;644(1-3):17-23. doi: 10.1016/j.ejphar.2010.06.042., Epub 2010 Jun 30. PMID:20599907 doi:http://dx.doi.org/10.1016/j.ejphar.2010.06.042
4. ↑ ter Haar E, Coll JT, Austen DA, Hsiao HM, Swenson L, Jain J. Structure of GSK3beta reveals a primed phosphorylation mechanism. Nat Struct Biol. 2001 Jul;8(7):593-6. PMID:11427888 doi:10.1038/89624
5. ↑ ter Haar E, Coll JT, Austen DA, Hsiao HM, Swenson L, Jain J. Structure of GSK3beta reveals a primed phosphorylation mechanism. Nat Struct Biol. 2001 Jul;8(7):593-6. PMID:11427888 doi:10.1038/89624
6. ↑ ter Haar E, Coll JT, Austen DA, Hsiao HM, Swenson L, Jain J. Structure of GSK3beta reveals a primed phosphorylation mechanism. Nat Struct Biol. 2001 Jul;8(7):593-6. PMID:11427888 doi:10.1038/89624
7. ↑ ^{7.0 7.1} Jeffrey, George A. An introduction to hydrogen bonding; Oxford University Press: Oxford, 1997
8. ↑ ^{8.0 8.1 8.2} Bertrand JA, Thieffine S, Vulpetti A, Cristiani C, Valsasina B, Knapp S, Kalisz HM, Flocco M. Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors. J Mol Biol. 2003 Oct 17;333(2):393-407. PMID:14529625