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Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

de la Mora-de la Mora I, Torres-Larios A, Enríquez-Flores S, Méndez ST, Castillo-Villanueva A, Gómez-Manzo S, López-Velázquez G, Marcial-Quino J, Torres-Arroyo A, García-Torres I, Reyes-Vivas H, Oria-Hernández J - PLoS ONE (2015)

Bottom Line: Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins.The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme.In contrast, the N15D mutant displays all the detrimental effects related to deamidation.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México, D.F., México.

ABSTRACT
Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

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The mutant N15D decrease the activity of HsTIM.The comparison between the wild type and the N15D mutant in the region of the catalytic residue K13 shows a different, inactive conformation. In all known TIMs, the structure of this region is conserved, even when compared to an enzyme from a bacterial source (TIM from Thermus thermophilus, PDB code 1YYA), which has a histidine residue in the equivalent position of HsTIM residue 15.
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pone.0123379.g010: The mutant N15D decrease the activity of HsTIM.The comparison between the wild type and the N15D mutant in the region of the catalytic residue K13 shows a different, inactive conformation. In all known TIMs, the structure of this region is conserved, even when compared to an enzyme from a bacterial source (TIM from Thermus thermophilus, PDB code 1YYA), which has a histidine residue in the equivalent position of HsTIM residue 15.

Mentions: Most importantly, the interface changes in the N15D mutant can be tracked to the active site of the mutated protein (Fig 10). In the N15D structure, the catalytic residue K13 moves from its canonical position out of the Ramachandran plot as in all TIM enzymes (phi/psi angles 51°/-148°, respectively) to an allowed conformation in the mutant (phi/psi angles -67°/-46°). The change can be clearly observed in the crystal structure as a displacement of 2.6 Å of the epsilon amino group of K13 (Fig 10). The result is consonant with the disrupted catalytic properties of the N15D mutant.


Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

de la Mora-de la Mora I, Torres-Larios A, Enríquez-Flores S, Méndez ST, Castillo-Villanueva A, Gómez-Manzo S, López-Velázquez G, Marcial-Quino J, Torres-Arroyo A, García-Torres I, Reyes-Vivas H, Oria-Hernández J - PLoS ONE (2015)

The mutant N15D decrease the activity of HsTIM.The comparison between the wild type and the N15D mutant in the region of the catalytic residue K13 shows a different, inactive conformation. In all known TIMs, the structure of this region is conserved, even when compared to an enzyme from a bacterial source (TIM from Thermus thermophilus, PDB code 1YYA), which has a histidine residue in the equivalent position of HsTIM residue 15.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401446&req=5

pone.0123379.g010: The mutant N15D decrease the activity of HsTIM.The comparison between the wild type and the N15D mutant in the region of the catalytic residue K13 shows a different, inactive conformation. In all known TIMs, the structure of this region is conserved, even when compared to an enzyme from a bacterial source (TIM from Thermus thermophilus, PDB code 1YYA), which has a histidine residue in the equivalent position of HsTIM residue 15.
Mentions: Most importantly, the interface changes in the N15D mutant can be tracked to the active site of the mutated protein (Fig 10). In the N15D structure, the catalytic residue K13 moves from its canonical position out of the Ramachandran plot as in all TIM enzymes (phi/psi angles 51°/-148°, respectively) to an allowed conformation in the mutant (phi/psi angles -67°/-46°). The change can be clearly observed in the crystal structure as a displacement of 2.6 Å of the epsilon amino group of K13 (Fig 10). The result is consonant with the disrupted catalytic properties of the N15D mutant.

Bottom Line: Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins.The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme.In contrast, the N15D mutant displays all the detrimental effects related to deamidation.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México, D.F., México.

ABSTRACT
Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

Show MeSH
Related in: MedlinePlus