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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.(2) 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.(1) 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

Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase monomer and has an overall structure that is characteristic of the apoenzyme. It has two phosphorylation sites that are involved in catalysis. One of these sites is Ser9, which is the phosphorylation site for AKT, resulting in the inactivation of GSK-3beta. The second phosphorylation site is Tyr216, located on the activation loop (shown in purple), and is responsible for the increase in catalytic activity. GSK-3beta has the characteristic two-domain kinase fold, containing a N-terminal beta-strand domain (red, residues 25-138) and a C-terminal alpha-helical domain (green, residues 139-343). There is an interface between the alpha and beta 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 (orange, residues 344-382) that are outside the main kinase fold. These residues form a small domain that closely packs next to the alpha-helical domain. The beta-strand domain is formed by seven beta-strands that run in an antiparallel formation. Strands 2-6 form a beta-barrel, through which a short alpha helix (yellow, residues 96-102) aligns against the beta-barrel. Two of this short helix’s residues play important roles in the catalytic action of GSK-3beta. A salt bridge, which is important in binding interactions, is positioned in the active site between Glu97 and Lys85. [4].

Binding Interactions

1)Folding of protein

a) location and description of position of alpha and beta sheets (Show in separate color on green screen)

b) crystal structure data (Table 1, Structural Characterizaiton of the GSK-3B Active Site Using Selective and Non-selective ATP-mimetic Inhibitors)

2)Substrate binding

a) “phosphate-binding” pocket: describe three crucial basic residues (Show in different color on green screen)

b) GSK-3 substrates and binding specifics- S/TxxxS/T (S=Serine, T=threonine, X= any amino acid)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217193/

http://www.rcsb.org/pdb/explore/explore.do?pdbId=1Q3D

Additional Features

The GSK-3β and staurosporine complex shows . It is observed that there are direct H-bonds, water-mediated polar interactions and hydrophobic interactions in the GSK-3β and staurosporine complex. 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 (cite Jeffrey). Since many pdb files lack hydrogen atoms, a significant H-bond can be considered when donor-acceptor distance are probably 3.5 Å (cite Jeffrey). 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 [cite J,A,Bertrand] 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 [cite J,A,Bertrand]. 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.

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 Alpha helices and Beta sheets on the protein as well as the complexed staurosporene molecule.

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) Bertrand, J. A., Thieffine, S., Vulpetti, A., et al. GSK-3 Beta complexed with Staurosporine 2003 (2) Capra, M , Nuciforo, P Confalonieri, S Quarto, S Bianchi, M Nebuloni, M Boldorini, R Pallotti, F. Frequent Alterations in the Expression of Serine/Threonine Kinases in Human Cancers 2006

(3) Hua F1, 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. 2010

(4) Haar, E., Coll, J. T., Austen, D. A., Hsiao, H-M., Swenson, L., Jain, J. Structure of GSK3B reveals a primed phosphorylation mechanism. Vertex Pharmaceuticals Incoprporated, Cambridge MA. Nature Publishing Group, Vol 8 No 7. July 2001.