E. coli MGMT Suicide Protein

Ben Canniff '19 and David Anderson '19

Contents:

I. Introduction

The Escherichia coli Ada gene O6 –methylgunine-DNA methyltransferace (MGMT) is a DNA binding protein that is involved in repairing mutations that occur during DNA replication. MGMT participates in methylation, which means it is a protein that adds a methyl group. MGMT adds a methyl group onto a specific cysteine residue, Cys146 , by breaking an ether bond connecting the methyl to the O6 guanine within the DNA chain. By simultaneously binding the methyl group onto itself and breaking the methyl's old bond, MGMT is rendered permanently inactive which is why it has earned the title of "The Suicide Protein".

The repair protein is located in the nucleus to allow it to have the best access to the newly replicated DNA. The alkyl groups targeted by MGMT are direct results from alkylating mutagens and carcinogens that cause mistakes in the replicated DNA. These mistakes must be fixed before the DNA replicates creating a mutant daughter strand.

 

(L., Stephanie, and Timothy R 2013)

II. General Structure

The suicidal DNA repair protein Ada O-6-methylguanine-DNA methyltransferase (MGMT) is 354 amino acids in length and 39 kDa in molecular mass. Ada is unique in the fact that it is present as a monomer in both its native and methylation state. The protein is predominantly composed of alpha helixes along with a group of beta sheets (Moore et al. 1996).

MGMT’s structure allows for stoichiometric alkyltransferase action at two different substrates. The action at different substrates is able to occur because MGMT contains two different functional domains, the 10 kDa N-terminal and the 19 kDa C-terminal (Moore et al. 1996).

The N-terminal domain acts as a phosphotriester-DNA, methyltansferase to accept alkyl groups from the diastereoisomers of alkylphosphotriester residues. The C-terminal domain, termed Ada-C, accepts alkyl groups at from O6 alkylguanine and O4 alkylthymines. The alpha helixes suggest a "helix-turn-helix" motif within the C-terminal Domain where MGMT contacts DNA. The Escherichia coli MGMT protein contains the Pro-Cys-His -Arg active site sequence contained in an alpha helix that is shared between all known organisms possessing an MGMT protein (Moore et al. 1996).

III. DNA Binding

MGMT’s native structure does not allow for the active site to interact with the target O6 methyl group. In order for the protein to correct a mutation on the DNA strand, it swivels one of the C-terminal helices to expose a possible DNA binding site. The swiveling of the C-terminal breaks the His147 and Glu 173 hydrogen bond allowing the His147 to rotate so it, along with the rest of the active site, faces the incoming DNA molecule. (Bhattacharyya et al. 1998) Cys 146 is the active site amino acid that performs a methytransferase action by while the helix-turn-helix motif contacts the major groove of the DNA. (Moore et al. 1996)

Cys 146 carries out a necleophilic attack on the target methyl group , which is allowed by a conformational change of the protein when a methylguanine is detected. This conformational change is required in order for MGMT to activate. Alkylation slightly distorts the phosphate-sugar backbone of dsDNA while MGMT shows a strong preference for dsDNA over ssDNA. The C-terminal active site helix attaches to the major groove of the DNA, covering around 8 base pairs of DNA. MGMT does not repair methylguanine in Z-DNA because its left hand helical orientation does not allow for MGMT to contact the DNA correctly (Reinhard et al. 2001). Z-DNA

Overview of DNA Binding

IV. Methy Transfer Reaction

The MGMT protein removes mutagenic methyl groups from guanines and thymines through the methyltransferase activity. MGMT binds the methyls to the sulfur molecule in Cys-146 located within the C-terminal. In order to transfer the methyl from the O6-methylguanine to the Cys-146, the protein must undergo acid catalyzed SN2 chemistry, which allows MGMT to bind the methyl while simultaneously cleaving the ether bond. While SN2 chemistry takes place a thiolate, an organosulfur compound, acts as a nucleophile. During the reaction a naturally associated positive charge from MGMT transfers to the O6 guanine and the carbon center undergoes a nucleophilic attack by the thiolate, thereby breaking the O 6 guanine’s ether bond to the DNA and binding it to the thiolate, forming a thioether, and the deactivated Cys-146 residue. Once the thioether bond is made the protein undergoes ubiquitination and is degraded. (Moore et al. 1996)

 

VI. References

Moore, M.h., J.m. Gulbis, E.j. Dodson, B. Demple, and P.c.e. Moody. "Ada O6-Methylguanine-Dna Methyltransferase From Escherichia Coli." (1996): n. pag. Web.

Bhattacharyya, Debasish, Tapas K. Hazra, W. David Behnke, Parkson L.-G. Chong, Alexander Kurosky, J. Ching Lee, and Sankar Mitra. "Reversible Folding of Ada Protein ( O 6 -Methylguanine?DNA Methyltransferase) of Escherichia Coli †." Biochemistry 37.6 (1998): 1722-730. Web.

Reinhard, Jost, William E. Hull, Claus-Wilhelm Von Der Lieth, Uta Eichhorn, Hans-Christian Kliem, Bernd Kaina, and Manfred Wiessler. "Monosaccharide-Linked Inhibitors OfO6-Methylguanine-DNA Methyltransferase (MGMT):  Synthesis, Molecular Modeling, and Structure?Activity Relationships." Journal of Medicinal Chemistry 44.24 (2001): 4050-061. Web.

L., Stephanie, and Timothy R. "Direct Repair in Mammalian Cells." New Research Directions in DNA Repair (2013): n. pag. Web.

Silva, Nathan, and David Marcey. "An Introduction to Jmol* Scripting** Nathan Silva and David Marcey © 2016." Intro to Jmol Scripting. N.p., 2016. Web. 07 Dec. 2016.

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