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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 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

-GSK-3beta has the typical two-domain kinase fold with a beta strand domain at the N-terminal end and an alpha-helical domain at the C-terminal end

-The ATP-binding site is at the interface of the alpha-helical and beta-strand domain and is bordered by the glycine-rich loop and the hinge

-The activation loop runs along the surface of the substrate binding groove

-The C-terminal residues are outside the core kinase fold and form a small domain that packs against the alpha-helical domain

-The beta-strand domain consists of seven antiparallel beta-strands

-Two phosphorylation sites that influence the catalytic activity of the protein

Haar, Ernst ter., Coll, Joyce T., Austen, Douglas A., Hsiao, Hsun-Mei., Swenson, Lora., Jain, Jugnu. Structure of GSK3B reveals a primed phosphorylation mechanism. Vertex Pharmaceuticals Incoprporated, Cambridge MA. Nature Publishing Group, Vol 8 No 7. July 2001.

-Monomer

-Overall structures of the enzyme are similar to that of the apoenzyme

-The crystals contain two molecules of GSK-3beta in the asymmetric unit and the two molecules are treated as identical

-Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine protein kinase

-Both isoforms have a conserved N-terminal serine reside (S21 for alpha and S9 for beta)

-Phosphorylation of the N-terminal serine residue plays an important role for further activity

Mishra, Nibha et al. Structure based virtual screening of GSK-3beta: Importance of protein flexibility and induced fit, 2009. Bioorganic & Medicinal Chemistry Letters, 2009, Vol. 19 Iss. 19, pp. 5582-5585. Retreived from http://www.sciencedirect.com/science/article/pii/S0960894X09011780

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 unique hydrogen bonding interactions compared to the other protein-staurosporine complexes. 

It is observed that there are direct hydrogen bonds, water-mediated polar interactions and hydrophobic interactions in the GSK-3β and staurosporine complex. There are only two direct hydrogen bonds, and they are observed between 1) the carbonyl oxygen of Asp 133 and N1 (nitrogen) of staurosporine. The length of this hydrogen bond is 2.93Å, 2) the backbone nitrogen of Val 135 and O5 (oxygen) of staurosporine. The length of this hydrogen bond is 2.76Å.

              The water-mediated polar interactions are between the carbonyl oxygen of Gln 185 and N4 (nitrogen) of the glycosidic ring.  

The length between these atoms is 4.47Å. The typical H bond is categorized to be between 2.2 and 4.0 Å (cite Jeffrey). Since many pdb files lack hydrogen atoms, one could classify a hydrogen bond between donor and acceptors that are 3.5Å apart. This interaction is outside all hydrogen bond classifications, and is a water mediated polar interaction between Gln 185 and glycosidic ring. 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 hydrogen bond interaction between two moieties. An example to this direct interaction can be observed in the AMP-PNP complex. Thr138 makes hydrogen bonds with 1) the backbone nitrogen of Arg 141 and 2)conserved water molecule.

              There is a significant number of  hydrophobic  interactions in the GSK-3β and staurosporine complex; to be more specific,  this complex buries  891 Å2 surface area. 

The hydrophobic residues that contribute to this surface are shown in pink in the green scene.

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 with respect to the two isoforms?

These green screens may help you.

Answer: At the N and C terminal lobes.

J.A. Bertrand, S. Thieffine, A. Vulpetti, C. Cristiani, B. Valsasina, S. Knapp, H.M. Kalisz, M. Flocco, Structural Characterization of the GSK-3β Active Site Using Selective and Non-selective ATP-mimetic Inhibitors, Journal of Molecular Biology, Volume 333, Issue 2, 17 October 2003, Pages 393-407, ISSN 0022-2836, http://dx.doi.org/10.1016/j.jmb.2003.08.031.

(http://www.sciencedirect.com/science/article/pii/S0022283603010593)

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