User:Ramiro Barrantes/FpgNeiRepair

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Contents

The FpgNei Protein Superfamily

Functional Units

G. Stereothermophilus Fpg E. Coli Nei

PDB ID 1R2Y

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PDB ID 1K3W

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Functional Cluster Variant 1 Variant 2 Fpg1 Fpg2 Plant Neil1 Neil2 Neil3 Proteo Actino1 Actino2 MimiVirus
Support for perfectly conserved Asn168 Y Y N N N N N N N N
Stability of catalytic helix Y Y Y N N N N N N N
Stability of intercalation loop Y Y Y N N N N N N Y
Intercalation loop Y Y Y N N N N N N Y
Zinc Finger Y Y N N Y Y Y Y Y N
Recognition complex none Y N N N N N N N N N
Neil1-specific: Support for Lys60 Y Y Y N Y Y Y Y Y Y
PlantFungi-specific: R254 DNA binding different in plants Y Y N Y Y Y N Y Y Y

Functional Cluster: Stability of perfectly conserved Asn168

WebLogo for the first functional unit, note the covariation between lysine nad arginine, which support the crucial Asn174
WebLogo for the first functional unit, note the covariation between lysine nad arginine, which support the crucial Asn174
Asn74, along with two other amino acids have an effect in the orientation and kinking of the DNA. In 4 of the 9 clades (Fpg1, Fpg2 and Plants and Fungi) Asn174 is supported by Lys160, which in turn hydrogen bonds with Leu249 and Ser250. In the other clades (Actinobacteria 1 and 2, Proteacteria and all vertebrate sequences), Arg171 that comes from a different helix fulfills the same roles as Lys160. One important difference is that the Zinc Finger is shaped differently in the absence of DNA, and there is a hydrogen bond between one of the beta-sheets and the arginine. One hypothesis is that the arginine or the lysine is necessary to support the Asn174, crucial for orientation of the DNA.

Functional Cluster: Stability of catalytic helix

The LER triad interact in stabilize the catalytic helix (Pro2 and Glu3), it is not clear how this is achieved in the other subfamilies
The LER triad interact in stabilize the catalytic helix (Pro2 and Glu3), it is not clear how this is achieved in the other subfamilies
The triad Leu4, Glu8 and Arg57 interact and provide stability to helixA, which has the catalytic residue Pro2,Glu3 and Glu6. This triad is present in the same four clades as above (Fpg1, Fpg2 and Plants and Fungi). This triad is not present in the remaining clades and it is not clear how the same stability is provided. Leu211 also has a hydrophobic interaction with Leu4.

Functional Cluster: Stability of intercalation loop

Image:IntercalationLoopSupport.jpg
This structure provides stability for the amino acids that insert into the space vacated by the damaged base
The intercalation loop inserts into the DNA when the base is everted. This structure, as well as a group of amino acids that support it exhibit high conservation in Fpg, AY, plants and Neil1, but not on the rest. Note that the amino acid composition suggests that mimivirus has this structure.

Functional Cluster: Stability of key Gly59 and Lys60

Notice the covariation in Neil1 and the rest with positions 134 and 137 and 170
Notice the covariation in Neil1 and the rest with positions 134 and 137 and 170
Gly59 and Lys60 are important in the activity of MutM. We hypothesize that Glu137 is very important to maintain its stability, this amino acid is compensated by Asn172 in Neil1 </ref>

Functional Cluster: Intercalation Loop

Image:IntercalationLoop.jpg
The residue in positions 77 and 78 suggest a possible intercalation loop
The intercalation loop inserts into the space left by the excised base.

The residue E2 and E6 have been mutated, with the first one inactivating the protein and the second one having no major effect [1].

Functional Cluster: Zinc/zincless finger

The Zinc Finger helps as a support of the key R264 residue
The Zinc Finger helps as a support of the key R264 residue
The Zinc finger (of the CCCC type) serves to hold the absolutely conserved Arg264 residue [2], which binds to the phosphate of the damaged base. The mutation of R252 results in failed cleavage of the damaged base [3]. Site directed mutagenesis experiments have been performed on all cysteines of this In Neil1, there is no Zinc but there is an equivalent structure [4]. Both the plants and mimivirus have a zincless finger, although it is not clear if this one is homologous

Functional Cluster: Recognition Complex

This complex is key in recognizing a damaged guanine[5]

Functional Cluster: DNA binding Tyrosine

Tyrosine that binds to DNA, the plant has a different structure alltogether
Tyrosine that binds to DNA, the plant has a different structure alltogether

Zinc/zincless finger

Tyrosine that binds to DNA, the plant has a different structure alltogether
Tyrosine that binds to DNA, the plant has a different structure alltogether

Evolution

The FpgNei evolution has not been easy to resolve [6], especially in the deeper branches. Assuming that functional clusters evolve more slowly than individual residues, we can use this as phylogenetic characters to 1) draw the most parsimonious evolution of the superfamily as dictated by these functional clusters 2) examine how these clusters have evolved and how this might have influenced the evolution of FpgNei. Image:Scenario1.png

Site-directed mutants

Name Organism/Structure Effect Reference
Escherichia coli (1k3w) inactive [7][8]
Escherichia coli (1k82) reduced glygosylase, less efficient in Schiff-base complex. [9][10]
Escherichia coli (1k3w) inactive, although can cleave AP sites [11]
Escherichia coli (1k3w) Inactive but can still make Schiff Base. [12]
Escherichia coli (1k3w) Decrease glycosylase, lyase ok. [13]
Escherichia coli (1k3w) Decrease glycosylase, lyase ok. [14]
Homo Sapiens (1tdh) Low activity [15]
E. Coli (1k82) No binding nor cleavage [16]
E. Coli (1k82) No binding nor cleavage. No zinc, no altered secondary structure [17]
E. Coli (1k82) No lyase nor glycosylase [18]

See structure column for structure reference

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References

  1. ↑ Burgess S, Jaruga P, Dodson ML, Dizdaroglu M, Lloyd RS. Determination of active site residues in Escherichia coli endonuclease VIII. J Biol Chem. 2002 Jan 25;277(4):2938-44. Epub 2001 Nov 15. PMID:11711552 doi:10.1074/jbc.M110499200
  2. ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
  3. ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
  4. ↑ Doublie S, Bandaru V, Bond JP, Wallace SS. The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc Natl Acad Sci U S A. 2004 Jul 13;101(28):10284-9. Epub 2004 Jul 1. PMID:15232006 doi:10.1073/pnas.0402051101
  5. ↑ Fromme JC, Verdine GL. DNA lesion recognition by the bacterial repair enzyme MutM. J Biol Chem. 2003 Dec 19;278(51):51543-8. Epub 2003 Oct 1. PMID:14525999 doi:10.1074/jbc.M307768200
  6. ↑ Doublie S, Bandaru V, Bond JP, Wallace SS. The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc Natl Acad Sci U S A. 2004 Jul 13;101(28):10284-9. Epub 2004 Jul 1. PMID:15232006 doi:10.1073/pnas.0402051101
  7. ↑ Golan G, Zharkov DO, Feinberg H, Fernandes AS, Zaika EI, Kycia JH, Grollman AP, Shoham G. Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility. Nucleic Acids Res. 2005 Sep 6;33(15):5006-16. Print 2005. PMID:16145054 doi:http://dx.doi.org/33/15/5006
  8. ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
  9. ↑ Sidorkina OM, Laval J. Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein). Nucleic Acids Res. 1998 Dec 1;26(23):5351-7. PMID:9826758
  10. ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
  11. ↑ Golan G, Zharkov DO, Feinberg H, Fernandes AS, Zaika EI, Kycia JH, Grollman AP, Shoham G. Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility. Nucleic Acids Res. 2005 Sep 6;33(15):5006-16. Print 2005. PMID:16145054 doi:http://dx.doi.org/33/15/5006
  12. ↑ Burgess S, Jaruga P, Dodson ML, Dizdaroglu M, Lloyd RS. Determination of active site residues in Escherichia coli endonuclease VIII. J Biol Chem. 2002 Jan 25;277(4):2938-44. Epub 2001 Nov 15. PMID:11711552 doi:10.1074/jbc.M110499200
  13. ↑ Burgess S, Jaruga P, Dodson ML, Dizdaroglu M, Lloyd RS. Determination of active site residues in Escherichia coli endonuclease VIII. J Biol Chem. 2002 Jan 25;277(4):2938-44. Epub 2001 Nov 15. PMID:11711552 doi:10.1074/jbc.M110499200
  14. ↑ Burgess S, Jaruga P, Dodson ML, Dizdaroglu M, Lloyd RS. Determination of active site residues in Escherichia coli endonuclease VIII. J Biol Chem. 2002 Jan 25;277(4):2938-44. Epub 2001 Nov 15. PMID:11711552 doi:10.1074/jbc.M110499200
  15. ↑ Shinmura K, Tao H, Goto M, Igarashi H, Taniguchi T, Maekawa M, Takezaki T, Sugimura H. Inactivating mutations of the human base excision repair gene NEIL1 in gastric cancer. Carcinogenesis. 2004 Dec;25(12):2311-7. Epub 2004 Aug 19. PMID:15319300 doi:10.1093/carcin/bgh267
  16. ↑ Tchou J, Michaels ML, Miller JH, Grollman AP. Function of the zinc finger in Escherichia coli Fpg protein. J Biol Chem. 1993 Dec 15;268(35):26738-44. PMID:8253809
  17. ↑ Tchou J, Michaels ML, Miller JH, Grollman AP. Function of the zinc finger in Escherichia coli Fpg protein. J Biol Chem. 1993 Dec 15;268(35):26738-44. PMID:8253809
  18. ↑ O'Connor TR, Graves RJ, de Murcia G, Castaing B, Laval J. Fpg protein of Escherichia coli is a zinc finger protein whose cysteine residues have a structural and/or functional role. J Biol Chem. 1993 Apr 25;268(12):9063-70. PMID:8473347

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Ramiro Barrantes, Jaime Prilusky

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