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pH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

Ciulli A, Lobley CM, Tuck KL, Smith AG, Blundell TL, Abell C - Acta Crystallogr. D Biol. Crystallogr. (2007)

Bottom Line: The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket.Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH.This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

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Affiliation: University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, England.

ABSTRACT
The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

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Crystal structure of the KPR–2′P-ADP-ribose binary complex and comparison with the KPR–NADP+ complex. (a) 2′P-ADP-ribose electron density. The final 2Fo − Fc electron-density map for 2′P-ADP-ribose contoured at 1σ is shown in blue. Binding modes of (b) 2′P-ADP-ribose and (c) NADP+ and the key residues at the active site of KPR are shown. The van der Waals surface of the N-terminal domain (residues 1–176) only is shown, coloured by electrostatic potential (neutral, white; positive, blue; negative, red). (d) Superposition of the KPR–NADP+ (salmon pink) and KPR–2′P-ADP-ribose (yellow) structures. The protein structures were superposed using the backbone atoms of residues 2–291.
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fig1: Crystal structure of the KPR–2′P-ADP-ribose binary complex and comparison with the KPR–NADP+ complex. (a) 2′P-ADP-ribose electron density. The final 2Fo − Fc electron-density map for 2′P-ADP-ribose contoured at 1σ is shown in blue. Binding modes of (b) 2′P-ADP-ribose and (c) NADP+ and the key residues at the active site of KPR are shown. The van der Waals surface of the N-terminal domain (residues 1–176) only is shown, coloured by electrostatic potential (neutral, white; positive, blue; negative, red). (d) Superposition of the KPR–NADP+ (salmon pink) and KPR–2′P-ADP-ribose (yellow) structures. The protein structures were superposed using the backbone atoms of residues 2–291.

Mentions: In an attempt to crystallize the ternary complex between KPR, NADPH and pantoate, we explored conditions similar to those used for the KPR–NADP+ crystals (Lobley et al., 2005 ▶) and obtained crystals at pH 4.0–5.0. Molecular replace­ment using AMoRe (Collaborative Computational Project, Number 4, 1994 ▶; Navaza, 1994 ▶, 2001 ▶) with apo KPR (Matak-Vinkovic et al., 2001 ▶) as the probe structure gave a solution with an Rcryst of 21.2% and a correlation coefficient of 55.7%. Initial 2Fo − Fc and Fo − Fc electron-density maps clearly showed the presence of a nonprotein molecule in the active site. However, no electron density corresponding to pantoate was identified; instead, it was possible to fit the density corresponding to part of NADPH. Ultimately, one molecule of 2′P-ADP-ribose was built into this density (Fig. 1 ▶a). Although electron density is incomplete for the terminal ribose, the presence of final 2Fo − Fc electron density at 3–5σ around the phosphate groups gave a clear indication of the position of the ligand and enabled its unambiguous modelling. Maximum-likelihood refinement implemented in REFMAC5 (Murshudov et al., 1997 ▶) was used to improve the quality of the electron-density maps and facilitate further rebuilding and improvement of the molecular model until no unexplained electron density remained and the Rcryst and Rfree values converged at 16.6 and 19.4%, respectively (Table 1 ▶). As in the apo (Matak-Vinkovic et al., 2001 ▶) and holo (Lobley et al., 2005 ▶) structures, the C-terminal residues were disordered and nine residues were not rebuilt.


pH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

Ciulli A, Lobley CM, Tuck KL, Smith AG, Blundell TL, Abell C - Acta Crystallogr. D Biol. Crystallogr. (2007)

Crystal structure of the KPR–2′P-ADP-ribose binary complex and comparison with the KPR–NADP+ complex. (a) 2′P-ADP-ribose electron density. The final 2Fo − Fc electron-density map for 2′P-ADP-ribose contoured at 1σ is shown in blue. Binding modes of (b) 2′P-ADP-ribose and (c) NADP+ and the key residues at the active site of KPR are shown. The van der Waals surface of the N-terminal domain (residues 1–176) only is shown, coloured by electrostatic potential (neutral, white; positive, blue; negative, red). (d) Superposition of the KPR–NADP+ (salmon pink) and KPR–2′P-ADP-ribose (yellow) structures. The protein structures were superposed using the backbone atoms of residues 2–291.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483484&req=5

fig1: Crystal structure of the KPR–2′P-ADP-ribose binary complex and comparison with the KPR–NADP+ complex. (a) 2′P-ADP-ribose electron density. The final 2Fo − Fc electron-density map for 2′P-ADP-ribose contoured at 1σ is shown in blue. Binding modes of (b) 2′P-ADP-ribose and (c) NADP+ and the key residues at the active site of KPR are shown. The van der Waals surface of the N-terminal domain (residues 1–176) only is shown, coloured by electrostatic potential (neutral, white; positive, blue; negative, red). (d) Superposition of the KPR–NADP+ (salmon pink) and KPR–2′P-ADP-ribose (yellow) structures. The protein structures were superposed using the backbone atoms of residues 2–291.
Mentions: In an attempt to crystallize the ternary complex between KPR, NADPH and pantoate, we explored conditions similar to those used for the KPR–NADP+ crystals (Lobley et al., 2005 ▶) and obtained crystals at pH 4.0–5.0. Molecular replace­ment using AMoRe (Collaborative Computational Project, Number 4, 1994 ▶; Navaza, 1994 ▶, 2001 ▶) with apo KPR (Matak-Vinkovic et al., 2001 ▶) as the probe structure gave a solution with an Rcryst of 21.2% and a correlation coefficient of 55.7%. Initial 2Fo − Fc and Fo − Fc electron-density maps clearly showed the presence of a nonprotein molecule in the active site. However, no electron density corresponding to pantoate was identified; instead, it was possible to fit the density corresponding to part of NADPH. Ultimately, one molecule of 2′P-ADP-ribose was built into this density (Fig. 1 ▶a). Although electron density is incomplete for the terminal ribose, the presence of final 2Fo − Fc electron density at 3–5σ around the phosphate groups gave a clear indication of the position of the ligand and enabled its unambiguous modelling. Maximum-likelihood refinement implemented in REFMAC5 (Murshudov et al., 1997 ▶) was used to improve the quality of the electron-density maps and facilitate further rebuilding and improvement of the molecular model until no unexplained electron density remained and the Rcryst and Rfree values converged at 16.6 and 19.4%, respectively (Table 1 ▶). As in the apo (Matak-Vinkovic et al., 2001 ▶) and holo (Lobley et al., 2005 ▶) structures, the C-terminal residues were disordered and nine residues were not rebuilt.

Bottom Line: The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket.Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH.This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, England.

ABSTRACT
The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

Show MeSH
Related in: MedlinePlus