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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 dimeric interface is highly perturbed in the structure of the N15D mutant.Panels A and C correspond to the WT HsTIM, whereas B and D correspond to the mutant N15D. (A) Hydrogen bonds in the interface of the wild-type enzyme that are lost in the N15D mutant (see S1 Table). The interactions were calculated with the HBplot server (http://dept.phy.bme.hu/virtuadrug/hbplot/bin/infopage.php). (B) There is a new intersubunit hydrogen bond in the N15D mutant that is not present in the wild type enzyme. This interaction is made between the mutated residue 15D and the side chain of S79. (C and D) Surface of a monomer interacting with the neighboring protein subunit. The total contact area per residue decreases from 2486 Å2 in the wild type enzyme (C) to 1561 Å2 on the N15D mutant (D) (see S2 Table), with the greatest decrease represented by residue M14 (red in C). These panels were drawn according to the results of the Contact Map Analysis server (http://ligin.weizmann.ac.il/cma/), which were plotted on the monomer surface according to the contact area of each residue on the interface on a blue (0 Å2) to red (211.8 Å2 for M14) scale basis.
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pone.0123379.g009: The dimeric interface is highly perturbed in the structure of the N15D mutant.Panels A and C correspond to the WT HsTIM, whereas B and D correspond to the mutant N15D. (A) Hydrogen bonds in the interface of the wild-type enzyme that are lost in the N15D mutant (see S1 Table). The interactions were calculated with the HBplot server (http://dept.phy.bme.hu/virtuadrug/hbplot/bin/infopage.php). (B) There is a new intersubunit hydrogen bond in the N15D mutant that is not present in the wild type enzyme. This interaction is made between the mutated residue 15D and the side chain of S79. (C and D) Surface of a monomer interacting with the neighboring protein subunit. The total contact area per residue decreases from 2486 Å2 in the wild type enzyme (C) to 1561 Å2 on the N15D mutant (D) (see S2 Table), with the greatest decrease represented by residue M14 (red in C). These panels were drawn according to the results of the Contact Map Analysis server (http://ligin.weizmann.ac.il/cma/), which were plotted on the monomer surface according to the contact area of each residue on the interface on a blue (0 Å2) to red (211.8 Å2 for M14) scale basis.

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 dimeric interface is highly perturbed in the structure of the N15D mutant.Panels A and C correspond to the WT HsTIM, whereas B and D correspond to the mutant N15D. (A) Hydrogen bonds in the interface of the wild-type enzyme that are lost in the N15D mutant (see S1 Table). The interactions were calculated with the HBplot server (http://dept.phy.bme.hu/virtuadrug/hbplot/bin/infopage.php). (B) There is a new intersubunit hydrogen bond in the N15D mutant that is not present in the wild type enzyme. This interaction is made between the mutated residue 15D and the side chain of S79. (C and D) Surface of a monomer interacting with the neighboring protein subunit. The total contact area per residue decreases from 2486 Å2 in the wild type enzyme (C) to 1561 Å2 on the N15D mutant (D) (see S2 Table), with the greatest decrease represented by residue M14 (red in C). These panels were drawn according to the results of the Contact Map Analysis server (http://ligin.weizmann.ac.il/cma/), which were plotted on the monomer surface according to the contact area of each residue on the interface on a blue (0 Å2) to red (211.8 Å2 for M14) scale basis.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g009: The dimeric interface is highly perturbed in the structure of the N15D mutant.Panels A and C correspond to the WT HsTIM, whereas B and D correspond to the mutant N15D. (A) Hydrogen bonds in the interface of the wild-type enzyme that are lost in the N15D mutant (see S1 Table). The interactions were calculated with the HBplot server (http://dept.phy.bme.hu/virtuadrug/hbplot/bin/infopage.php). (B) There is a new intersubunit hydrogen bond in the N15D mutant that is not present in the wild type enzyme. This interaction is made between the mutated residue 15D and the side chain of S79. (C and D) Surface of a monomer interacting with the neighboring protein subunit. The total contact area per residue decreases from 2486 Å2 in the wild type enzyme (C) to 1561 Å2 on the N15D mutant (D) (see S2 Table), with the greatest decrease represented by residue M14 (red in C). These panels were drawn according to the results of the Contact Map Analysis server (http://ligin.weizmann.ac.il/cma/), which were plotted on the monomer surface according to the contact area of each residue on the interface on a blue (0 Å2) to red (211.8 Å2 for M14) scale basis.
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