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PDBsum entry 1eh6

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protein metals links
Transferase PDB id
1eh6
Jmol PyMol
Contents
Protein chain
168 a.a. *
Metals
_ZN
Waters ×153
* Residue conservation analysis
PDB id:
1eh6
Name: Transferase
Title: Human o6-alkylguanine-DNA alkyltransferase
Structure: O6-alkylguanine-DNA alkyltransferase. Chain: a. Synonym: agt, o6-methylguanine-DNA methyltransferase, mgmt. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562
Resolution:
2.00Å     R-factor:   0.197     R-free:   0.218
Authors: D.S.Daniels,J.A.Tainer
Key ref:
D.S.Daniels et al. (2000). Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. EMBO J, 19, 1719-1730. PubMed id: 10747039 DOI: 10.1093/emboj/19.7.1719
Date:
18-Feb-00     Release date:   12-Apr-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P16455  (MGMT_HUMAN) -  Methylated-DNA--protein-cysteine methyltransferase
Seq:
Struc:
207 a.a.
168 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.2.1.1.63  - Methylated-DNA--[protein]-cysteine S-methyltransferase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: DNA (containing 6-O-methylguanine) + protein L-cysteine = DNA (without 6-O-methylguanine) + protein S-methyl-L-cysteine
DNA (containing 6-O-methylguanine)
+ protein L-cysteine
= DNA (without 6-O-methylguanine)
+ protein S-methyl-L-cysteine
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   3 terms 
  Biological process     cellular response to organic cyclic compound   20 terms 
  Biochemical function     catalytic activity     8 terms  

 

 
    Added reference    
 
 
DOI no: 10.1093/emboj/19.7.1719 EMBO J 19:1719-1730 (2000)
PubMed id: 10747039  
 
 
Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding.
D.S.Daniels, C.D.Mol, A.S.Arvai, S.Kanugula, A.E.Pegg, J.A.Tainer.
 
  ABSTRACT  
 
Human O(6)-alkylguanine-DNA alkyltransferase (AGT), which directly reverses endogenous alkylation at the O(6)-position of guanine, confers resistance to alkylation chemotherapies and is therefore an active anticancer drug target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes reveal an unexpected zinc-stabilized helical bridge joining a two-domain alpha/beta structure. An asparagine hinge couples the active site motif to a helix-turn-helix (HTH) motif implicated in DNA binding. The reactive cysteine environment, its position within a groove adjacent to the alkyl-binding cavity and mutational analyses characterize DNA-damage recognition and inhibitor specificity, support a structure-based dealkylation mechanism and suggest a molecular basis for destabilization of the alkylated protein. These results support damaged nucleotide flipping facilitated by an arginine finger within the HTH motif to stabilize the extrahelical O(6)-alkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cysteine alkylation sterically shifts the HTH recognition helix to evidently mechanistically couple release of repaired DNA to an opening of the protein fold to promote the biological turnover of the alkylated protein.
 
  Selected figure(s)  
 
Figure 1.
Figure 1 Human AGT secondary structure, two-domain fold, zinc site, and location of structurally and catalytically critical residues. (A) Overall fold, displaying the active site cysteine (yellow) and its surrounding hydrogen-bond network, zinc (purple) ligands and Arg128 'arginine finger'. A central interdomain cleft separates the N-terminal / roll ( -helices in royal blue, -strands in orange) from the C-terminal domain, which contains the active site and HTH DNA-binding motif (sky blue). For continuity, the location of the internal disordered loop (purple) has been interpolated. (B) Primary sequence, secondary structure and residue function of human AGT aligned with the E.coli Ada-C protein. Identity between the two sequences is shown with an asterisk, while corresponding residues that lie within 4.5 Å in the overlaid structures are given in upper case letters. The conserved active site motif (yellow boxes) is flanked by residues defining the O^6-alkylguanine-binding channel (green boxes), anticipated DNA-binding residues (orange boxes), and HTH motif, with its associated Arg128 (sky blue). Colors distinguish residues participating in the active site hydrogen-bond network (red), zinc ligands (purple), -helices (royal blue) and 3[10]-helices (navy blue).
Figure 4.
Figure 4 Structurally inferred guanine- and DNA-binding mode of AGT. (A) Guanine inserts into the active site channel along the recognition helix, stacking the aromatic base against Met134 and Gly131. Guanine-specific hydrogen bonds occur between the Watson–Crick base-pairing atoms of guanine and protein main chain atoms. The benzyl lesion is positioned adjacent to the active site cysteine, and oriented for optimal S[N]2 displacement. (B) Overlay of AGT and the HTH-containing DNA-binding region of the catabolite gene activator protein bound to DNA (both red), and proposed analogous binding of DNA (purple) by AGT. Arg128, extending from the HTH motif (sky blue), penetrates the base stack and is ideally positioned to facilitate flipping of target O^6-alkylguanine nucleotides. (C) Potential DNA-contacting residues of AGT (same color scheme as for Figure 1A), rotated by 90° about the vertical axis relative to (B). The HTH motif (sky blue), lying within the major groove, provides several potential hydrophilic and electrostatic DNA contacts. The adjacent 'wing', corresponding to the turn between B6 and B7, contacts the minor groove through Ser151 and Ser152. (D) The electrostatic surface of AGT, oriented as (C) and colored by Coulombic electrostatic potential (kT/e^-), displays a positively charged region centered around Arg128 complementary to the DNA phosphate backbone (oxygens, red; phosphates, yellow). Arg128, the 'arginine finger', facilitates damage repair by entering the base stack and displacing the extrahelical O^6-alkylguanine nucleotide.
 
  The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2000, 19, 1719-1730) copyright 2000.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20714665 F.P.McManus, Q.Fang, J.D.Booth, A.M.Noronha, A.E.Pegg, and C.J.Wilds (2010).
Synthesis and characterization of an O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand cross-link in a 5'-GNC motif and repair by human O(6)-alkylguanine-DNA alkyltransferase.
  Org Biomol Chem, 8, 4414-4426.  
20502938 J.L.Tubbs, and J.A.Tainer (2010).
Alkyltransferase-like proteins: molecular switches between DNA repair pathways.
  Cell Mol Life Sci, 67, 3749-3762.  
  20871819 L.A.Peterson (2010).
Formation, repair, and genotoxic properties of bulky DNA adducts formed from tobacco-specific nitrosamines.
  J Nucleic Acids, 2010, 0.  
20026607 Q.Fang, S.Kanugula, J.L.Tubbs, J.A.Tainer, and A.E.Pegg (2010).
Repair of O4-alkylthymine by O6-alkylguanine-DNA alkyltransferases.
  J Biol Chem, 285, 8185-8195.  
20412404 S.H.Huang, P.Y.Chang, C.J.Liu, M.W.Lin, and K.T.Hsia (2010).
O6-methylguanine-DNA methyltransferase gene coding region polymorphisms and oral cancer risk.
  J Oral Pathol Med, 39, 645-650.  
  20371487 W.Wei, B.Li, M.A.Hanes, S.Kakar, X.Chen, and L.Liu (2010).
S-nitrosylation from GSNOR deficiency impairs DNA repair and promotes hepatocarcinogenesis.
  Sci Transl Med, 2, 19ra13.  
19472322 A.G.Kalapila, N.A.Loktionova, and A.E.Pegg (2009).
Effect of O6-alkylguanine-DNA alkyltransferase on genotoxicity of epihalohydrins.
  Environ Mol Mutagen, 50, 502-514.  
19358853 C.A.Adams, M.Melikishvili, D.W.Rodgers, J.J.Rasimas, A.E.Pegg, and M.G.Fried (2009).
Topologies of complexes containing O6-alkylguanine-DNA alkyltransferase and DNA.
  J Mol Biol, 389, 248-263.  
19516334 J.L.Tubbs, V.Latypov, S.Kanugula, A.Butt, M.Melikishvili, R.Kraehenbuehl, O.Fleck, A.Marriott, A.J.Watson, B.Verbeek, G.McGown, M.Thorncroft, M.F.Santibanez-Koref, C.Millington, A.S.Arvai, M.D.Kroeger, L.A.Peterson, D.M.Williams, M.G.Fried, G.P.Margison, A.E.Pegg, and J.A.Tainer (2009).
Flipping of alkylated DNA damage bridges base and nucleotide excision repair.
  Nature, 459, 808-813.
PDB codes: 3gva 3gx4 3gyh
19531487 R.Guza, L.Ma, Q.Fang, A.E.Pegg, and N.Tretyakova (2009).
Cytosine methylation effects on the repair of O6-methylguanines within CG dinucleotides.
  J Biol Chem, 284, 22601-22610.  
18712882 A.G.Kalapila, N.A.Loktionova, and A.E.Pegg (2008).
Alkyltransferase-mediated toxicity of 1,3-butadiene diepoxide.
  Chem Res Toxicol, 21, 1851-1861.  
18973327 G.T.Pauly, N.A.Loktionova, Q.Fang, S.L.Vankayala, W.C.Guida, and A.E.Pegg (2008).
Substitution of aminomethyl at the meta-position enhances the inactivation of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine.
  J Med Chem, 51, 7144-7153.  
18353991 J.Hu, A.Ma, and A.R.Dinner (2008).
A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT.
  Proc Natl Acad Sci U S A, 105, 4615-4620.  
19055796 P.Slama, I.Filippis, and M.Lappe (2008).
Detection of protein catalytic residues at high precision using local network properties.
  BMC Bioinformatics, 9, 517.  
18803403 Q.Fang, A.M.Noronha, S.P.Murphy, C.J.Wilds, J.L.Tubbs, J.A.Tainer, G.Chowdhury, F.P.Guengerich, and A.E.Pegg (2008).
Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase.
  Biochemistry, 47, 10892-10903.  
17996846 Q.Fang, N.A.Loktionova, R.C.Moschel, S.Javanmard, G.T.Pauly, and A.E.Pegg (2008).
Differential inactivation of polymorphic variants of human O6-alkylguanine-DNA alkyltransferase.
  Biochem Pharmacol, 75, 618-626.  
18033718 S.Banala, A.Arnold, and K.Johnsson (2008).
Caged substrates for protein labeling and immobilization.
  Chembiochem, 9, 38-41.  
17482892 A.E.Pegg, Q.Fang, and N.A.Loktionova (2007).
Human variants of O6-alkylguanine-DNA alkyltransferase.
  DNA Repair (Amst), 6, 1071-1078.  
17485252 J.L.Tubbs, A.E.Pegg, and J.A.Tainer (2007).
DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy.
  DNA Repair (Amst), 6, 1100-1115.  
17376731 J.Penner-Hahn (2007).
Zinc-promoted alkyl transfer: a new role for zinc.
  Curr Opin Chem Biol, 11, 166-171.  
17485251 R.J.Hansen, S.M.Ludeman, S.J.Paikoff, A.E.Pegg, and M.E.Dolan (2007).
Role of MGMT in protecting against cyclophosphamide-induced toxicity in cells and animals.
  DNA Repair (Amst), 6, 1145-1154.  
17880193 S.Javanmard, N.A.Loktionova, Q.Fang, G.T.Pauly, A.E.Pegg, and R.C.Moschel (2007).
Inactivation of O(6)-alkylguanine-DNA alkyltransferase by folate esters of O(6)-benzyl-2'-deoxyguanosine and of O(6)-[4-(hydroxymethyl)benzyl]guanine.
  J Med Chem, 50, 5193-5201.  
16633564 A.R.Hornillo-Araujo, A.J.Burrell, M.K.Aiertza, T.Shibata, D.M.Hammond, D.Edmont, H.Adams, G.P.Margison, and D.M.Williams (2006).
The syntheses and properties of tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine and O6-methylguanine.
  Org Biomol Chem, 4, 1723-1729.  
16826543 A.Roberts, J.G.Pelton, and D.E.Wemmer (2006).
Structural studies of MJ1529, an O6-methylguanine-DNA methyltransferase.
  Magn Reson Chem, 44, S71-S82.
PDB code: 2g7h
16698182 C.A.Rabik, M.C.Njoku, and M.E.Dolan (2006).
Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy.
  Cancer Treat Rev, 32, 261-276.  
16464003 Y.Mishina, E.M.Duguid, and C.He (2006).
Direct reversal of DNA alkylation damage.
  Chem Rev, 106, 215-232.  
15934048 A.Juillerat, C.Heinis, I.Sielaff, J.Barnikow, H.Jaccard, B.Kunz, A.Terskikh, and K.Johnsson (2005).
Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells.
  Chembiochem, 6, 1263-1269.  
16160825 S.K.Kufer, H.Dietz, C.Albrecht, K.Blank, A.Kardinal, M.Rief, and H.E.Gaub (2005).
Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy.
  Eur Biophys J, 35, 72-78.  
15731349 S.Kanugula, G.T.Pauly, R.C.Moschel, and A.E.Pegg (2005).
A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities.
  Proc Natl Acad Sci U S A, 102, 3617-3622.  
15221026 D.S.Daniels, T.T.Woo, K.X.Luu, D.M.Noll, N.D.Clarke, A.E.Pegg, and J.A.Tainer (2004).
DNA binding and nucleotide flipping by the human DNA repair protein AGT.
  Nat Struct Mol Biol, 11, 714-720.
PDB codes: 1t38 1t39
14691244 J.J.Rasimas, P.A.Dalessio, I.J.Ropson, A.E.Pegg, and M.G.Fried (2004).
Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase.
  Protein Sci, 13, 301-305.  
15057289 S.L.Gerson (2004).
MGMT: its role in cancer aetiology and cancer therapeutics.
  Nat Rev Cancer, 4, 296-307.  
15280878 T.J.Begley, and L.D.Samson (2004).
Reversing DNA damage with a directional bias.
  Nat Struct Mol Biol, 11, 688-690.  
12725859 A.Juillerat, T.Gronemeyer, A.Keppler, S.Gendreizig, H.Pick, H.Vogel, and K.Johnsson (2003).
Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo.
  Chem Biol, 10, 313-317.  
14522053 E.M.Duguid, Y.Mishina, and C.He (2003).
How do DNA repair proteins locate potential base lesions? a chemical crosslinking method to investigate O6-alkylguanine-DNA alkyltransferases.
  Chem Biol, 10, 827-835.  
12496275 J.J.Rasimas, A.E.Pegg, and M.G.Fried (2003).
DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability.
  J Biol Chem, 278, 7973-7980.  
12151404 L.Liu, A.E.Pegg, K.M.Williams, and F.P.Guengerich (2002).
Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.
  J Biol Chem, 277, 37920-37928.  
11564893 A.K.Teo, H.K.Oh, R.B.Ali, and B.F.Li (2001).
The modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase is a negative regulator of estrogen receptor-mediated transcription upon alkylation DNA damage.
  Mol Cell Biol, 21, 7105-7114.  
  11574685 D.M.Noll, and N.D.Clarke (2001).
Covalent capture of a human O(6)-alkylguanine alkyltransferase-DNA complex using N(1),O(6)-ethanoxanthosine, a mechanism-based crosslinker.
  Nucleic Acids Res, 29, 4025-4034.  
11732917 J.C.Delaney, and J.M.Essigmann (2001).
Effect of sequence context on O(6)-methylguanine repair and replication in vivo.
  Biochemistry, 40, 14968-14975.  
11746760 S.Kanugula, and A.E.Pegg (2001).
Novel DNA repair alkyltransferase from Caenorhabditis elegans.
  Environ Mol Mutagen, 38, 235-243.  
11557810 X.Cheng, and R.J.Roberts (2001).
AdoMet-dependent methylation, DNA methyltransferases and base flipping.
  Nucleic Acids Res, 29, 3784-3795.  
11000262 P.E.Verdemato, J.A.Brannigan, C.Damblon, F.Zuccotto, P.C.Moody, and L.Y.Lian (2000).
DNA-binding mechanism of the Escherichia coli Ada O(6)-alkylguanine-DNA alkyltransferase.
  Nucleic Acids Res, 28, 3710-3718.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.

 

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