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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 N15D mutant is solely responsible for the structural changes induced by deamidation in HsTIM.(A) The enzyme dimers of the wild type and the N15D mutant do not superpose. Superposition of the dimers of WT HsTIM (cyan) and the N15D mutant (green). The monomers are closer to each other in wild-type TIM when compared to the N15D mutant enzyme. If this superposition is made using a monomer as a reference, the angle of association is changed by 14°, as defined by the DynDom server (http://fizz.cmp.uea.ac.uk/dyndom/). (B) The monomeric subunits superpose well. If only the protein monomers are superimposed, the overall RMSD value on Cα is 0.6 Å (see S5 Fig). There are two main regions that change their overall conformation, loop 1 (in magenta, RMSD of 3 Å) and loop 3 (in red, RMSD of 4 Å). (C) The N71D mutation has no structural effects on the enzyme. A closer look on the region of loop 1 and loop 3 shows that, whereas the N15D mutation (in magenta and red) undergoes a conformational change in both regions, the N71D mutation (in light red) keeps the structure of the WT HsTIM. (D) Residue 15 on the mutant N15D makes two new interactions. A novel intersubunit interaction is made between the side chains of D15 and S79. Also, an intrasubunit interaction is made with the side chain of R17. In the wild-type enzyme, R17 makes intersubunit interactions with the side chains of T70 and N71 (see Fig 9).
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pone.0123379.g008: The N15D mutant is solely responsible for the structural changes induced by deamidation in HsTIM.(A) The enzyme dimers of the wild type and the N15D mutant do not superpose. Superposition of the dimers of WT HsTIM (cyan) and the N15D mutant (green). The monomers are closer to each other in wild-type TIM when compared to the N15D mutant enzyme. If this superposition is made using a monomer as a reference, the angle of association is changed by 14°, as defined by the DynDom server (http://fizz.cmp.uea.ac.uk/dyndom/). (B) The monomeric subunits superpose well. If only the protein monomers are superimposed, the overall RMSD value on Cα is 0.6 Å (see S5 Fig). There are two main regions that change their overall conformation, loop 1 (in magenta, RMSD of 3 Å) and loop 3 (in red, RMSD of 4 Å). (C) The N71D mutation has no structural effects on the enzyme. A closer look on the region of loop 1 and loop 3 shows that, whereas the N15D mutation (in magenta and red) undergoes a conformational change in both regions, the N71D mutation (in light red) keeps the structure of the WT HsTIM. (D) Residue 15 on the mutant N15D makes two new interactions. A novel intersubunit interaction is made between the side chains of D15 and S79. Also, an intrasubunit interaction is made with the side chain of R17. In the wild-type enzyme, R17 makes intersubunit interactions with the side chains of T70 and N71 (see Fig 9).

Mentions: The comparison between the crystal structures of the WT HsTIM (PDB code 2JK2) and the N71D mutant shows that both structures are highly similar (S4A Fig, Fig 8C), with a Cα RMSD of 0.26 Å; the lateral chain of D71 is in the same conformation as the parental N71 (S4B Fig, Fig 8C). There are minimal discernible changes in the region of the mutation; in the B subunit, the N15 sidechain is now found in a double conformation (S4C Fig, Fig 8C). One conformation shows the same position as that in the WT structure, whereas in the alternate conformation the sidechain moves slightly towards the mutated D71 residue (S4B Fig, Fig 8C). This local change does not alter the structure in the vicinity of the mutation (S4B Fig, Fig 8C), nor does it affect the geometry of the active site (S4D Fig). The results are consistent with the mild effects of the N71D mutation on the structure and function of HsTIM. In contrast, the crystal structure of the N15D mutant provides a clear atomic description of the conformational changes explaining the detrimental effects of deamidation on HsTIM. The superposition of the WT and N15D structures shows that deamidation of N15 disturbs the assembly of the dimer (Fig 8A); the association between the monomers is changed in such a way that the angle between both subunits in the N15D structure is increased by 14°, with reference to the WT structure (Fig 8A). A close examination of the WT and N15D HsTIM monomers indicates that loop 1 and loop 3 (residues 13 to 16 and 69 to 76, respectively), both critical components of the interface of TIMs, are altered in the mutant protein (Fig 8B and S5 Fig). In the crystal structure of this mutant, the lateral chain of the introduced D15 rotates from its original position in the WT structure (Fig 8, panels C-D) to establish a new salt bridge with the adjacent R17 of its own subunit and S79 of the adjacent monomer (Fig 8D). It is noteworthy that R17 has, in WT HsTIM, cross-subunit interactions with the side chains of T70 and N71 (Fig 9A), which are lost in the N15D mutant (Fig 9B). Overall, just these few new interactions of D15 (R17 and S79, Fig 8D) trigger the conformational change of loop 1 and the concomitant loss of interactions with loop 3, which finally provoke the perturbation of the HsTIM interface.


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 N15D mutant is solely responsible for the structural changes induced by deamidation in HsTIM.(A) The enzyme dimers of the wild type and the N15D mutant do not superpose. Superposition of the dimers of WT HsTIM (cyan) and the N15D mutant (green). The monomers are closer to each other in wild-type TIM when compared to the N15D mutant enzyme. If this superposition is made using a monomer as a reference, the angle of association is changed by 14°, as defined by the DynDom server (http://fizz.cmp.uea.ac.uk/dyndom/). (B) The monomeric subunits superpose well. If only the protein monomers are superimposed, the overall RMSD value on Cα is 0.6 Å (see S5 Fig). There are two main regions that change their overall conformation, loop 1 (in magenta, RMSD of 3 Å) and loop 3 (in red, RMSD of 4 Å). (C) The N71D mutation has no structural effects on the enzyme. A closer look on the region of loop 1 and loop 3 shows that, whereas the N15D mutation (in magenta and red) undergoes a conformational change in both regions, the N71D mutation (in light red) keeps the structure of the WT HsTIM. (D) Residue 15 on the mutant N15D makes two new interactions. A novel intersubunit interaction is made between the side chains of D15 and S79. Also, an intrasubunit interaction is made with the side chain of R17. In the wild-type enzyme, R17 makes intersubunit interactions with the side chains of T70 and N71 (see Fig 9).
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Related In: Results  -  Collection

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pone.0123379.g008: The N15D mutant is solely responsible for the structural changes induced by deamidation in HsTIM.(A) The enzyme dimers of the wild type and the N15D mutant do not superpose. Superposition of the dimers of WT HsTIM (cyan) and the N15D mutant (green). The monomers are closer to each other in wild-type TIM when compared to the N15D mutant enzyme. If this superposition is made using a monomer as a reference, the angle of association is changed by 14°, as defined by the DynDom server (http://fizz.cmp.uea.ac.uk/dyndom/). (B) The monomeric subunits superpose well. If only the protein monomers are superimposed, the overall RMSD value on Cα is 0.6 Å (see S5 Fig). There are two main regions that change their overall conformation, loop 1 (in magenta, RMSD of 3 Å) and loop 3 (in red, RMSD of 4 Å). (C) The N71D mutation has no structural effects on the enzyme. A closer look on the region of loop 1 and loop 3 shows that, whereas the N15D mutation (in magenta and red) undergoes a conformational change in both regions, the N71D mutation (in light red) keeps the structure of the WT HsTIM. (D) Residue 15 on the mutant N15D makes two new interactions. A novel intersubunit interaction is made between the side chains of D15 and S79. Also, an intrasubunit interaction is made with the side chain of R17. In the wild-type enzyme, R17 makes intersubunit interactions with the side chains of T70 and N71 (see Fig 9).
Mentions: The comparison between the crystal structures of the WT HsTIM (PDB code 2JK2) and the N71D mutant shows that both structures are highly similar (S4A Fig, Fig 8C), with a Cα RMSD of 0.26 Å; the lateral chain of D71 is in the same conformation as the parental N71 (S4B Fig, Fig 8C). There are minimal discernible changes in the region of the mutation; in the B subunit, the N15 sidechain is now found in a double conformation (S4C Fig, Fig 8C). One conformation shows the same position as that in the WT structure, whereas in the alternate conformation the sidechain moves slightly towards the mutated D71 residue (S4B Fig, Fig 8C). This local change does not alter the structure in the vicinity of the mutation (S4B Fig, Fig 8C), nor does it affect the geometry of the active site (S4D Fig). The results are consistent with the mild effects of the N71D mutation on the structure and function of HsTIM. In contrast, the crystal structure of the N15D mutant provides a clear atomic description of the conformational changes explaining the detrimental effects of deamidation on HsTIM. The superposition of the WT and N15D structures shows that deamidation of N15 disturbs the assembly of the dimer (Fig 8A); the association between the monomers is changed in such a way that the angle between both subunits in the N15D structure is increased by 14°, with reference to the WT structure (Fig 8A). A close examination of the WT and N15D HsTIM monomers indicates that loop 1 and loop 3 (residues 13 to 16 and 69 to 76, respectively), both critical components of the interface of TIMs, are altered in the mutant protein (Fig 8B and S5 Fig). In the crystal structure of this mutant, the lateral chain of the introduced D15 rotates from its original position in the WT structure (Fig 8, panels C-D) to establish a new salt bridge with the adjacent R17 of its own subunit and S79 of the adjacent monomer (Fig 8D). It is noteworthy that R17 has, in WT HsTIM, cross-subunit interactions with the side chains of T70 and N71 (Fig 9A), which are lost in the N15D mutant (Fig 9B). Overall, just these few new interactions of D15 (R17 and S79, Fig 8D) trigger the conformational change of loop 1 and the concomitant loss of interactions with loop 3, which finally provoke the perturbation of the HsTIM interface.

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