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The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions.

Ruan J, Mouveaux T, Light SH, Minasov G, Anderson WF, Tomavo S, Ngô HM - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays.Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development.Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Structural Genomics of Infectious Diseases, Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611, USA.

ABSTRACT
In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

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The TgENO1 active site. (a) Superposition of the TgENO1 dimer (green) with the ‘open-loop’ ScENO dimer (blue; PDB entry 1ebh; Wedekind et al., 1995 ▶). The r.m.s.d. is 0.56 Å over 777 Cα atoms. (b) Superposition of the TgENO1 dimer with the ‘closed-loop’ hENO1 dimer (orange; PDB entry 3b97; Kang et al., 2008 ▶). The r.m.s.d. is 0.68 Å over 645 Cα atoms. (c) A different perspective of the superposition in (a) highlights the hydrogen bond (dashed line) between the unique TgENO1 residue Glu164 in loop 2 and residue Tyr270 in loop 3. (d) Superposition of TgENO1 on the closed-loop PEP-bound complex (PDB entry 3ucd; Qin et al., 2012 ▶) illustrates that L2 closure necessitates breakage of the Glu164–Tyr270 hydrogen bond and should allow His165 to hydrogen-bond to a PEP O atom.
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fig4: The TgENO1 active site. (a) Superposition of the TgENO1 dimer (green) with the ‘open-loop’ ScENO dimer (blue; PDB entry 1ebh; Wedekind et al., 1995 ▶). The r.m.s.d. is 0.56 Å over 777 Cα atoms. (b) Superposition of the TgENO1 dimer with the ‘closed-loop’ hENO1 dimer (orange; PDB entry 3b97; Kang et al., 2008 ▶). The r.m.s.d. is 0.68 Å over 645 Cα atoms. (c) A different perspective of the superposition in (a) highlights the hydrogen bond (dashed line) between the unique TgENO1 residue Glu164 in loop 2 and residue Tyr270 in loop 3. (d) Superposition of TgENO1 on the closed-loop PEP-bound complex (PDB entry 3ucd; Qin et al., 2012 ▶) illustrates that L2 closure necessitates breakage of the Glu164–Tyr270 hydrogen bond and should allow His165 to hydrogen-bond to a PEP O atom.

Mentions: Conversion from the tachyzoite stage to the bradyzoite stage is accompanied by a dramatic shift from oxidative to anaerobic glycolytic processes (Dzierszinski et al., 2004 ▶; Tomavo, 2001 ▶). Potentially important in this transformation, bradyzoite TgENO1 exhibits a similar Km value for 2-phospho-d-glycerate when compared with the tachyzoite TgENO2, but has a threefold lower kcat (Dzierszinski et al., 1999 ▶). The enolase active site is located in the central cavity formed by the C-termini of the β-strands and contains loop 1 (L1; residues 36–57), loop 2 (L2; residues 166–173) and loop 3 (L3; residues 258–282) (Figs. 1 ▶, 2 ▶ and 4 ▶). Previous studies have reported that these loops adopt an ‘open’ conformation in the unliganded and PEP-bound states, but undergo conformational changes to adopt a ‘closed’ conformation in 2-phospho-d-glycerate-bound and Mg2+-bound states (Lebioda et al., 1989 ▶; Zhang et al., 1997 ▶). L1 acts a ‘lid’ and closes the active site upon binding of substrate and cofactor using the residue Ser41/39 (TgENO1/human ENO1) in L1 by coordinating with the Mg2+ cofactor. The closed conformation facilitates protonation by His165/159 of the α carbon in 2-phospho-d-glycerate and the subsequent deprotonation by Lys355/345 to form a carbanion intermediate. Phosphoenolpyruvate is formed by elimination of hydroxide from carbon 3 by residue Glu217/211 and is then released as the three dynamic loops shift to an ‘open’ conformation. Superimposing TgENO1 with previously reported enolase structures shows the three loops in the apo TgENO1 structure assume an ‘open’ state (Figs. 4 ▶a and 4 ▶b).


The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions.

Ruan J, Mouveaux T, Light SH, Minasov G, Anderson WF, Tomavo S, Ngô HM - Acta Crystallogr. D Biol. Crystallogr. (2015)

The TgENO1 active site. (a) Superposition of the TgENO1 dimer (green) with the ‘open-loop’ ScENO dimer (blue; PDB entry 1ebh; Wedekind et al., 1995 ▶). The r.m.s.d. is 0.56 Å over 777 Cα atoms. (b) Superposition of the TgENO1 dimer with the ‘closed-loop’ hENO1 dimer (orange; PDB entry 3b97; Kang et al., 2008 ▶). The r.m.s.d. is 0.68 Å over 645 Cα atoms. (c) A different perspective of the superposition in (a) highlights the hydrogen bond (dashed line) between the unique TgENO1 residue Glu164 in loop 2 and residue Tyr270 in loop 3. (d) Superposition of TgENO1 on the closed-loop PEP-bound complex (PDB entry 3ucd; Qin et al., 2012 ▶) illustrates that L2 closure necessitates breakage of the Glu164–Tyr270 hydrogen bond and should allow His165 to hydrogen-bond to a PEP O atom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356359&req=5

fig4: The TgENO1 active site. (a) Superposition of the TgENO1 dimer (green) with the ‘open-loop’ ScENO dimer (blue; PDB entry 1ebh; Wedekind et al., 1995 ▶). The r.m.s.d. is 0.56 Å over 777 Cα atoms. (b) Superposition of the TgENO1 dimer with the ‘closed-loop’ hENO1 dimer (orange; PDB entry 3b97; Kang et al., 2008 ▶). The r.m.s.d. is 0.68 Å over 645 Cα atoms. (c) A different perspective of the superposition in (a) highlights the hydrogen bond (dashed line) between the unique TgENO1 residue Glu164 in loop 2 and residue Tyr270 in loop 3. (d) Superposition of TgENO1 on the closed-loop PEP-bound complex (PDB entry 3ucd; Qin et al., 2012 ▶) illustrates that L2 closure necessitates breakage of the Glu164–Tyr270 hydrogen bond and should allow His165 to hydrogen-bond to a PEP O atom.
Mentions: Conversion from the tachyzoite stage to the bradyzoite stage is accompanied by a dramatic shift from oxidative to anaerobic glycolytic processes (Dzierszinski et al., 2004 ▶; Tomavo, 2001 ▶). Potentially important in this transformation, bradyzoite TgENO1 exhibits a similar Km value for 2-phospho-d-glycerate when compared with the tachyzoite TgENO2, but has a threefold lower kcat (Dzierszinski et al., 1999 ▶). The enolase active site is located in the central cavity formed by the C-termini of the β-strands and contains loop 1 (L1; residues 36–57), loop 2 (L2; residues 166–173) and loop 3 (L3; residues 258–282) (Figs. 1 ▶, 2 ▶ and 4 ▶). Previous studies have reported that these loops adopt an ‘open’ conformation in the unliganded and PEP-bound states, but undergo conformational changes to adopt a ‘closed’ conformation in 2-phospho-d-glycerate-bound and Mg2+-bound states (Lebioda et al., 1989 ▶; Zhang et al., 1997 ▶). L1 acts a ‘lid’ and closes the active site upon binding of substrate and cofactor using the residue Ser41/39 (TgENO1/human ENO1) in L1 by coordinating with the Mg2+ cofactor. The closed conformation facilitates protonation by His165/159 of the α carbon in 2-phospho-d-glycerate and the subsequent deprotonation by Lys355/345 to form a carbanion intermediate. Phosphoenolpyruvate is formed by elimination of hydroxide from carbon 3 by residue Glu217/211 and is then released as the three dynamic loops shift to an ‘open’ conformation. Superimposing TgENO1 with previously reported enolase structures shows the three loops in the apo TgENO1 structure assume an ‘open’ state (Figs. 4 ▶a and 4 ▶b).

Bottom Line: Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays.Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development.Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Structural Genomics of Infectious Diseases, Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611, USA.

ABSTRACT
In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

Show MeSH
Related in: MedlinePlus