Limits...
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.

Show MeSH

Related in: MedlinePlus

Crystal structures of TvTIM1 and point mutants at residue I45.Surface and ribbon representation of the ball and socket interaction at the dimer interface. Monomer A is represented as ribbons and monomer B is represented as coil. Residues of the socket (F44, A62, V65, I82, F85 and I87) of monomer A are shown in blue in the left panel and gray in the right panel, the ball residue (position 45) of monomer B is depicted in dark magenta in both panels. In the left panel, the cavity that is formed by residues of the socket is observed in a sliced view of the hydrophobicity surface of monomer A with the ball residue 45 of monomer B inside the cavity. Mutant I45G (B) shows a water molecule inside the cavity at H-bond distance from residues 45 and 46 of monomer B and residue 81 of monomer A. Right panel, a closer view of residues of the socket and the ball is shown. TvTIM1 structure (A) shows the most common rotamer for the side-chain of residue I82, whereas I45G (B), I45A (C), I45V (D) and I45L (E) mutants present the second frequent rotamer. I45F and I45Y enzymes display the third frequent rotamer for I82 residue.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664265&req=5

pone.0141747.g006: Crystal structures of TvTIM1 and point mutants at residue I45.Surface and ribbon representation of the ball and socket interaction at the dimer interface. Monomer A is represented as ribbons and monomer B is represented as coil. Residues of the socket (F44, A62, V65, I82, F85 and I87) of monomer A are shown in blue in the left panel and gray in the right panel, the ball residue (position 45) of monomer B is depicted in dark magenta in both panels. In the left panel, the cavity that is formed by residues of the socket is observed in a sliced view of the hydrophobicity surface of monomer A with the ball residue 45 of monomer B inside the cavity. Mutant I45G (B) shows a water molecule inside the cavity at H-bond distance from residues 45 and 46 of monomer B and residue 81 of monomer A. Right panel, a closer view of residues of the socket and the ball is shown. TvTIM1 structure (A) shows the most common rotamer for the side-chain of residue I82, whereas I45G (B), I45A (C), I45V (D) and I45L (E) mutants present the second frequent rotamer. I45F and I45Y enzymes display the third frequent rotamer for I82 residue.

Mentions: Crystal structures of TvTIM1 mutants were solved by molecular replacement, all proteins crystallized in the P22121 space group with one molecule in the asymmetric unit that assembles as a dimer in the biologic unit of the crystal (S1 Table). The RMSD deviation in Cα-atom positions after superposition of mutants to the wild-type structure ranges from 0.13 to 0.24 Å with the exception of I45Y that shows a RMSD of 0.51 Å (Fig 6).


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)

Crystal structures of TvTIM1 and point mutants at residue I45.Surface and ribbon representation of the ball and socket interaction at the dimer interface. Monomer A is represented as ribbons and monomer B is represented as coil. Residues of the socket (F44, A62, V65, I82, F85 and I87) of monomer A are shown in blue in the left panel and gray in the right panel, the ball residue (position 45) of monomer B is depicted in dark magenta in both panels. In the left panel, the cavity that is formed by residues of the socket is observed in a sliced view of the hydrophobicity surface of monomer A with the ball residue 45 of monomer B inside the cavity. Mutant I45G (B) shows a water molecule inside the cavity at H-bond distance from residues 45 and 46 of monomer B and residue 81 of monomer A. Right panel, a closer view of residues of the socket and the ball is shown. TvTIM1 structure (A) shows the most common rotamer for the side-chain of residue I82, whereas I45G (B), I45A (C), I45V (D) and I45L (E) mutants present the second frequent rotamer. I45F and I45Y enzymes display the third frequent rotamer for I82 residue.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141747.g006: Crystal structures of TvTIM1 and point mutants at residue I45.Surface and ribbon representation of the ball and socket interaction at the dimer interface. Monomer A is represented as ribbons and monomer B is represented as coil. Residues of the socket (F44, A62, V65, I82, F85 and I87) of monomer A are shown in blue in the left panel and gray in the right panel, the ball residue (position 45) of monomer B is depicted in dark magenta in both panels. In the left panel, the cavity that is formed by residues of the socket is observed in a sliced view of the hydrophobicity surface of monomer A with the ball residue 45 of monomer B inside the cavity. Mutant I45G (B) shows a water molecule inside the cavity at H-bond distance from residues 45 and 46 of monomer B and residue 81 of monomer A. Right panel, a closer view of residues of the socket and the ball is shown. TvTIM1 structure (A) shows the most common rotamer for the side-chain of residue I82, whereas I45G (B), I45A (C), I45V (D) and I45L (E) mutants present the second frequent rotamer. I45F and I45Y enzymes display the third frequent rotamer for I82 residue.
Mentions: Crystal structures of TvTIM1 mutants were solved by molecular replacement, all proteins crystallized in the P22121 space group with one molecule in the asymmetric unit that assembles as a dimer in the biologic unit of the crystal (S1 Table). The RMSD deviation in Cα-atom positions after superposition of mutants to the wild-type structure ranges from 0.13 to 0.24 Å with the exception of I45Y that shows a RMSD of 0.51 Å (Fig 6).

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