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Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

Bottom Line: In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer.Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model.The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

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Comparison of the normalized B-factor values of TvTIM1 and mutants at residue 45.Crystal structure of the wild-type enzyme and the mutants ribbons indicating the variation of the normalized B-factors (A) TvTIM1 structure, the mean value of the normalized B-factor is represented in cyan, the lower value is blue, and the highest B-factor value is red. (B) I45G, (C) I45A, (D) I45V, (E) I45L, (F) I45F and (G) I45Y. The position of the eight α-helices of the (β-α)8 barrel fold is indicated by arrows (H1–H8) on TvTIM1 structure (A).
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pone.0141747.g007: Comparison of the normalized B-factor values of TvTIM1 and mutants at residue 45.Crystal structure of the wild-type enzyme and the mutants ribbons indicating the variation of the normalized B-factors (A) TvTIM1 structure, the mean value of the normalized B-factor is represented in cyan, the lower value is blue, and the highest B-factor value is red. (B) I45G, (C) I45A, (D) I45V, (E) I45L, (F) I45F and (G) I45Y. The position of the eight α-helices of the (β-α)8 barrel fold is indicated by arrows (H1–H8) on TvTIM1 structure (A).

Mentions: Another significant structural change at the residues in the hydrophobic cavity is present at residue F85. In the wild-type enzyme and TvTIM1 mutants with smaller side-chain residues at position 45, I85 delimits the exit of the hydrophobic cavity (Fig 6). As a result of the presence of a bulky residue at position 45 in mutant I45F, I85 side-chain is seen shifted away, by 4.61Å for the ξ carbon of F85 compared to the wild-type structure (Fig 6). A comparison of the normalized B-factors for TvTIM1 and residue 45 mutants indicates that this mutation did not increase flexibility/disorder in the neighbor residues (Fig 7).


Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

Comparison of the normalized B-factor values of TvTIM1 and mutants at residue 45.Crystal structure of the wild-type enzyme and the mutants ribbons indicating the variation of the normalized B-factors (A) TvTIM1 structure, the mean value of the normalized B-factor is represented in cyan, the lower value is blue, and the highest B-factor value is red. (B) I45G, (C) I45A, (D) I45V, (E) I45L, (F) I45F and (G) I45Y. The position of the eight α-helices of the (β-α)8 barrel fold is indicated by arrows (H1–H8) on TvTIM1 structure (A).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664265&req=5

pone.0141747.g007: Comparison of the normalized B-factor values of TvTIM1 and mutants at residue 45.Crystal structure of the wild-type enzyme and the mutants ribbons indicating the variation of the normalized B-factors (A) TvTIM1 structure, the mean value of the normalized B-factor is represented in cyan, the lower value is blue, and the highest B-factor value is red. (B) I45G, (C) I45A, (D) I45V, (E) I45L, (F) I45F and (G) I45Y. The position of the eight α-helices of the (β-α)8 barrel fold is indicated by arrows (H1–H8) on TvTIM1 structure (A).
Mentions: Another significant structural change at the residues in the hydrophobic cavity is present at residue F85. In the wild-type enzyme and TvTIM1 mutants with smaller side-chain residues at position 45, I85 delimits the exit of the hydrophobic cavity (Fig 6). As a result of the presence of a bulky residue at position 45 in mutant I45F, I85 side-chain is seen shifted away, by 4.61Å for the ξ carbon of F85 compared to the wild-type structure (Fig 6). A comparison of the normalized B-factors for TvTIM1 and residue 45 mutants indicates that this mutation did not increase flexibility/disorder in the neighbor residues (Fig 7).

Bottom Line: In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer.Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model.The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

Show MeSH
Related in: MedlinePlus