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Insights into the activity and specificity of Trypanosoma cruzi trans-sialidase from molecular dynamics simulations.

Mitchell FL, Neres J, Ramraj A, Raju RK, Hillier IH, Vincent MA, Bryce RA - Biochemistry (2013)

Bottom Line: These simulations indicate that, where lactose is released, this leaving group samples well-defined interactions in the acceptor site, some of which are mediated by localized water molecules; also, the extent of the opening of the acceptor site to solvent is reduced as compared with those of unliganded forms of TcTS.Thus, the predicted solvent accessibility of sialylated TcTS is increased when 4-methylumbelliferyl α-d-acetylneuraminide is the substrate compared to sialyllactose; this in turn is likely to contribute to a greater propensity for hydrolysis of the covalent intermediate.These computational simulations, which suggest that protein flexibility has a role in the transferase/sialidase activity of TcTS, have the potential to aid in the design of anti-Chagas inhibitors effective against this neglected tropical disease.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy and Pharmaceutical Sciences and ‡School of Chemistry, University of Manchester , Oxford Road, Manchester M13 9PT, U.K.

ABSTRACT
Trypanosoma cruzitrans-sialidase (TcTS), which catalyzes the transfer or hydrolysis of terminal sialic acid residues, is crucial to the development and proliferation of the T. cruzi parasite and thus has emerged as a potential drug target for the treatment of Chagas disease. We here probe the origin of the observed preference for the transfer reaction over hydrolysis where the substrate for TcTS is the natural sialyl donor (represented in this work by sialyllactose). Thus, acceptor lactose preferentially attacks the sialyl-enyzme intermediate rather than water. We compare this with the weaker preference for such transfer shown by a synthetic donor substrate, 4-methylumbelliferyl α-d-acetylneuraminide. For this reason, we conducted molecular dynamics simulations of TcTS following its sialylation by the substrate to examine the behavior of the asialyl leaving group by the protein. These simulations indicate that, where lactose is released, this leaving group samples well-defined interactions in the acceptor site, some of which are mediated by localized water molecules; also, the extent of the opening of the acceptor site to solvent is reduced as compared with those of unliganded forms of TcTS. However, where there is release of 4-methylumbelliferone, this leaving group explores a range of transient poses; surrounding active site water is also more disordered. The acceptor site explores more open conformations, similar to the case in which the 4-methylumbelliferone is absent. Thus, the predicted solvent accessibility of sialylated TcTS is increased when 4-methylumbelliferyl α-d-acetylneuraminide is the substrate compared to sialyllactose; this in turn is likely to contribute to a greater propensity for hydrolysis of the covalent intermediate. These computational simulations, which suggest that protein flexibility has a role in the transferase/sialidase activity of TcTS, have the potential to aid in the design of anti-Chagas inhibitors effective against this neglected tropical disease.

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Distances (inÅngstroms) between the centers of mass of sialylresidues of sialylated TcTS and (a) lactose or (b) 4-methylumbelliferoneduring three replicate MD simulations (black, light gray, and darkgray).
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fig5: Distances (inÅngstroms) between the centers of mass of sialylresidues of sialylated TcTS and (a) lactose or (b) 4-methylumbelliferoneduring three replicate MD simulations (black, light gray, and darkgray).

Mentions: We now consider the threereplicate MD simulations of sialyl-TcTS with lactose in the acceptorsite. During the majority of the simulations, lactose remains closeto the sialyl residue, with a short distance between the center ofmass of the TcTS-bound sialyl moiety and the lactose of ∼4Å (Figure 5a). This proximity corresponds,from inspection, to a well-defined bound pose in the acceptor site,where lactose stacks against Tyr119 and Trp312 (Figure 6). By virtue of this stacking interaction, Tyr119 remainsmainly in an outward conformation, with a χ1 valueof −60° (Figure S3e of the SupportingInformation). The cohesion of the Trp312–lactose–Tyr119motif is also illustrated by a low degree of fluctuation in the Trp312–Tyr119intercentroid distance, which remains at ∼9 Å (Figure 3e). This short distance suggests that a significantopening of the Tyr119/Trp312 acceptor site, which could have allowedgreater solvent exposure of the active site, does not occur.


Insights into the activity and specificity of Trypanosoma cruzi trans-sialidase from molecular dynamics simulations.

Mitchell FL, Neres J, Ramraj A, Raju RK, Hillier IH, Vincent MA, Bryce RA - Biochemistry (2013)

Distances (inÅngstroms) between the centers of mass of sialylresidues of sialylated TcTS and (a) lactose or (b) 4-methylumbelliferoneduring three replicate MD simulations (black, light gray, and darkgray).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675669&req=5

fig5: Distances (inÅngstroms) between the centers of mass of sialylresidues of sialylated TcTS and (a) lactose or (b) 4-methylumbelliferoneduring three replicate MD simulations (black, light gray, and darkgray).
Mentions: We now consider the threereplicate MD simulations of sialyl-TcTS with lactose in the acceptorsite. During the majority of the simulations, lactose remains closeto the sialyl residue, with a short distance between the center ofmass of the TcTS-bound sialyl moiety and the lactose of ∼4Å (Figure 5a). This proximity corresponds,from inspection, to a well-defined bound pose in the acceptor site,where lactose stacks against Tyr119 and Trp312 (Figure 6). By virtue of this stacking interaction, Tyr119 remainsmainly in an outward conformation, with a χ1 valueof −60° (Figure S3e of the SupportingInformation). The cohesion of the Trp312–lactose–Tyr119motif is also illustrated by a low degree of fluctuation in the Trp312–Tyr119intercentroid distance, which remains at ∼9 Å (Figure 3e). This short distance suggests that a significantopening of the Tyr119/Trp312 acceptor site, which could have allowedgreater solvent exposure of the active site, does not occur.

Bottom Line: These simulations indicate that, where lactose is released, this leaving group samples well-defined interactions in the acceptor site, some of which are mediated by localized water molecules; also, the extent of the opening of the acceptor site to solvent is reduced as compared with those of unliganded forms of TcTS.Thus, the predicted solvent accessibility of sialylated TcTS is increased when 4-methylumbelliferyl α-d-acetylneuraminide is the substrate compared to sialyllactose; this in turn is likely to contribute to a greater propensity for hydrolysis of the covalent intermediate.These computational simulations, which suggest that protein flexibility has a role in the transferase/sialidase activity of TcTS, have the potential to aid in the design of anti-Chagas inhibitors effective against this neglected tropical disease.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy and Pharmaceutical Sciences and ‡School of Chemistry, University of Manchester , Oxford Road, Manchester M13 9PT, U.K.

ABSTRACT
Trypanosoma cruzitrans-sialidase (TcTS), which catalyzes the transfer or hydrolysis of terminal sialic acid residues, is crucial to the development and proliferation of the T. cruzi parasite and thus has emerged as a potential drug target for the treatment of Chagas disease. We here probe the origin of the observed preference for the transfer reaction over hydrolysis where the substrate for TcTS is the natural sialyl donor (represented in this work by sialyllactose). Thus, acceptor lactose preferentially attacks the sialyl-enyzme intermediate rather than water. We compare this with the weaker preference for such transfer shown by a synthetic donor substrate, 4-methylumbelliferyl α-d-acetylneuraminide. For this reason, we conducted molecular dynamics simulations of TcTS following its sialylation by the substrate to examine the behavior of the asialyl leaving group by the protein. These simulations indicate that, where lactose is released, this leaving group samples well-defined interactions in the acceptor site, some of which are mediated by localized water molecules; also, the extent of the opening of the acceptor site to solvent is reduced as compared with those of unliganded forms of TcTS. However, where there is release of 4-methylumbelliferone, this leaving group explores a range of transient poses; surrounding active site water is also more disordered. The acceptor site explores more open conformations, similar to the case in which the 4-methylumbelliferone is absent. Thus, the predicted solvent accessibility of sialylated TcTS is increased when 4-methylumbelliferyl α-d-acetylneuraminide is the substrate compared to sialyllactose; this in turn is likely to contribute to a greater propensity for hydrolysis of the covalent intermediate. These computational simulations, which suggest that protein flexibility has a role in the transferase/sialidase activity of TcTS, have the potential to aid in the design of anti-Chagas inhibitors effective against this neglected tropical disease.

Show MeSH
Related in: MedlinePlus