User:Andrew Wills/Sandbox 1
From Proteopedia
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==Active Sites and Mechanism== | ==Active Sites and Mechanism== | ||
The HhH segment of AlkA connects to the phosphodiester backbone of DNA by hydrogen bonding and <scene name='56/566536/Sodium_ion_interaction/1'>sodium ion interaction</scene>. The binding of the HhH segment to DNA stabilizes the damaged base and creates a 66 degree bend away from the protein that widens the minor groove of DNA.<ref name="Moe">Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print. </ref> <scene name='56/566536/Leu_125/2'>Leu 125</scene> fits into the minor groove between base pairs and allows the alkylated base to be flipped into the active site.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> | The HhH segment of AlkA connects to the phosphodiester backbone of DNA by hydrogen bonding and <scene name='56/566536/Sodium_ion_interaction/1'>sodium ion interaction</scene>. The binding of the HhH segment to DNA stabilizes the damaged base and creates a 66 degree bend away from the protein that widens the minor groove of DNA.<ref name="Moe">Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print. </ref> <scene name='56/566536/Leu_125/2'>Leu 125</scene> fits into the minor groove between base pairs and allows the alkylated base to be flipped into the active site.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> | ||
| - | The AlkA active site is located where the second and third domains are separated by a deep nonpolar cleft that is lined with the aromatic side chains Phe18, Tyr273, Trp272, Tyr222, and Trp218.<ref name="Labahn">Labahn, Jorg, Orlando Scharer, et al. "Structural Basis for the Excision Repair of Alkylation-Damaged DNA." Cell. 86.2 (1996): 321-329. Print.</ref> These side chains create a <scene name='56/566536/Binding_pocket/1'>DNA Binding Pocket</scene> for the alkylated base.<ref name="Labahn">Labahn, Jorg, Orlando Scharer, et al. "Structural Basis for the Excision Repair of Alkylation-Damaged DNA." Cell. 86.2 (1996): 321-329. Print.</ref> Once the damaged base is in the active site, <scene name='56/566536/Trp_272/1'>Trp272</scene> stabilizes the flipped out alkylated base in the binding pocket by aromatic ring-stacking interactions.<ref name="Moe">Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print. </ref> <scene name='56/566536/Asp_238/1'>Asp238</scene> is essential for allowing the reaction to proceed, and points into the nonpolar pocket in order to allow stabilization of a carbocation intermediate. This stabilization is what allows the cleavage of the glycosidic bond on the damaged base.<ref name="Moe">Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print. </ref> | + | The AlkA active site is located where the second and third domains are separated by a deep nonpolar cleft that is lined with the aromatic side chains Phe18, Tyr273, Trp272, Tyr222, and Trp218.<ref name="Labahn">Labahn, Jorg, Orlando Scharer, et al. "Structural Basis for the Excision Repair of Alkylation-Damaged DNA." Cell. 86.2 (1996): 321-329. Print.</ref> These side chains create a <scene name='56/566536/Binding_pocket/1'>DNA Binding Pocket</scene> for the alkylated base.<ref name="Labahn">Labahn, Jorg, Orlando Scharer, et al. "Structural Basis for the Excision Repair of Alkylation-Damaged DNA." Cell. 86.2 (1996): 321-329. Print.</ref> Once the damaged base is in the active site, <scene name='56/566536/Trp_272/1'>Trp272</scene> stabilizes the flipped out alkylated base in the binding pocket by aromatic ring-stacking interactions.<ref name="Moe">Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print. </ref> <scene name='56/566536/Asp_238/1'>Asp238</scene> is essential for allowing the reaction to proceed, and points into the nonpolar pocket in order to allow stabilization of a carbocation intermediate. This stabilization is what allows the cleavage of the glycosidic bond on the damaged base.<ref name="Moe">Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print. </ref> |
==DNA Interaction== | ==DNA Interaction== | ||
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DNA is held in place by interactions with the HhH segment, which acts as a platform to create the 66 degree bend in DNA mentioned earlier. The bending of the DNA relieves tensional strain created by the widening of the minor groove for base flipping.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> Amide nitrogens on residues 210, 212, and 215 hydrogen bond with DNA’s phosphodiester backbone, while sodium metal ion interactions further stabilize DNA in the molecule.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> | DNA is held in place by interactions with the HhH segment, which acts as a platform to create the 66 degree bend in DNA mentioned earlier. The bending of the DNA relieves tensional strain created by the widening of the minor groove for base flipping.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> Amide nitrogens on residues 210, 212, and 215 hydrogen bond with DNA’s phosphodiester backbone, while sodium metal ion interactions further stabilize DNA in the molecule.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> | ||
| - | In order to keep the DNA strand bound to the protein, AlkA depends on van der Waals interactions on the minor groove of DNA. The van der Waals interactions play a major role in AlkA’s preference for double stranded DNA | + | In order to keep the DNA strand bound to the protein, AlkA depends on van der Waals interactions on the minor groove of DNA. The van der Waals interactions play a major role in AlkA’s preference for double stranded DNA.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> An example of these interactions may be seen with the Pro175 wedged into the minor groove of DNA and anchoring it by van der Waals interctions.<ref name="Hollis">Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print. </ref> |
| - | The essential Asp238 residue creates hydrogen bonds with the | + | The essential Asp238 residue creates hydrogen bonds with the alkylated DNA base that has been flipped into the active site. Its high affinity binding for the modified base holds it in the active site for cleavage of the glycosidic bond. An inhibitor or mutation to the Asp238 residue would ultimately prevent the cell's ability to modify DNA by base excision repair. |
Revision as of 20:27, 6 November 2013
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| 1diz, resolution 2.50Å () | |||||||||
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| Non-Standard Residues: | |||||||||
| Activity: | DNA-3-methyladenine glycosylase II, with EC number 3.2.2.21 | ||||||||
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| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
Contents |
CRYSTAL STRUCTURE OF E. COLI 3-METHYLADENINE DNA GLYCOSYLASE (ALKA) COMPLEXED WITH DNA
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General Function
1diz Escherichia coli 3 methyladenine DNA glycosylase II () is a DNA repair enzyme that initiates base excision repair for the removal of alkylated bases. There are many environmental toxins and cellular agents that may alkylate DNA bases. One example is Aflatoxin, a compound that is converted into a reactive epoxide by cytochrome P450 and attacks guanosine at its N-7 atom to form alkylated bases[1]. The alkylated DNA bases prevent regulatory proteins from binding to DNA and blocks replicative polymerases to stop DNA synthesis.[2] AlkA initiates base excision repair by first locating and binding to the alkylated DNA. It has broad substrate specificity and is able to remove a variety of different alkylated bases. AlkA then flips the affected base out of the DNA double helix and into the active site of the enzyme. Once in the active site, AlkA hydrolyzes the glycosidic bond to release the damaged base and leave the sugar phosphate backbone intact. This creates the AP site that is either devoid of a purine or pyridine. The AP site signals to other base excision repair enzymes to insert an undamaged nucleotide based on the undamaged complementary strand and seal the DNA.
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See Also
References
- ↑ Berg, Jeremy, Tymoczko John, and Lubert Stryer. Biochemistry. 6th. New York: W.H. Freeman and Company, 2007. 806-808. Print.
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Hollis, Thomas, Yoshitaka Ichikawa, and Tom Ellenberger. "DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA." EMBO Journal. 19.4 (2000): 758-766. Print.
- ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 Labahn, Jorg, Orlando Scharer, et al. "Structural Basis for the Excision Repair of Alkylation-Damaged DNA." Cell. 86.2 (1996): 321-329. Print.
- ↑ 4.0 4.1 4.2 4.3 Moe, E, D.R. Hall, et al. "Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans." Biological Crystallography. 68.6 (2012): 703-712. Print.
