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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.

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.

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Changes in protonation states tune the binding mode of 2′P-ADP-ribose. (a) At pH 7.7 both the 2′-phosphate group of the ligand and Glu256 will be negatively charged, carrying a −2 and a −1 charge, respectively. Their interaction would be highly unfavourable. The ligand binds in the same orientation observed for NADP+, anchoring the 2′-phosphate close to Arg31 and hydrogen bonding to Glu256 via the terminal ribose. (b) Binding mode observed in the KPR–2′P-ADP-ribose crystal structure reported here. At pH 4.5 the 2′-phosphate becomes protonated (pK = 6.5) carrying a single negative charge. Glu256 is likely to protonate, allowing a favourable hydrogen bond to the 2′-phosphate group.
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fig3: Changes in protonation states tune the binding mode of 2′P-ADP-ribose. (a) At pH 7.7 both the 2′-phosphate group of the ligand and Glu256 will be negatively charged, carrying a −2 and a −1 charge, respectively. Their interaction would be highly unfavourable. The ligand binds in the same orientation observed for NADP+, anchoring the 2′-phosphate close to Arg31 and hydrogen bonding to Glu256 via the terminal ribose. (b) Binding mode observed in the KPR–2′P-ADP-ribose crystal structure reported here. At pH 4.5 the 2′-phosphate becomes protonated (pK = 6.5) carrying a single negative charge. Glu256 is likely to protonate, allowing a favourable hydrogen bond to the 2′-phosphate group.

Mentions: We interpret these data as showing that the binding mode of 2′P-ADP-ribose is reversed at pH 4.5, presumably as a consequence of changes in protonation states (Fig. 3 ▶). The second pK of the 2′-phosphate group will be around 6.4 (Mas & Colman, 1984 ▶). At pH 7.7 the 2′-phosphate will be doubly charged and will interact strongly with Arg31, whereas interaction with the carboxylate side chain of Glu256, which is also negatively charged, would be unfavourable (Fig. 3 ▶a). In contrast, at pH 4.5 the 2′-phosphate would be present as the monoanion and Glu256 will be partially protonated. Interaction with the 2′-phosphate of the ligand will then be more favourable (Fig. 3 ▶b) and is observed in the crystal structure at a hydrogen-bonding distance of 2.4 Å. These changes in protonation states have triggered an otherwise unfavourable contact between a carboxylate and phosphate group and contributed significantly to the reversed binding mode observed in the crystal.


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)

Changes in protonation states tune the binding mode of 2′P-ADP-ribose. (a) At pH 7.7 both the 2′-phosphate group of the ligand and Glu256 will be negatively charged, carrying a −2 and a −1 charge, respectively. Their interaction would be highly unfavourable. The ligand binds in the same orientation observed for NADP+, anchoring the 2′-phosphate close to Arg31 and hydrogen bonding to Glu256 via the terminal ribose. (b) Binding mode observed in the KPR–2′P-ADP-ribose crystal structure reported here. At pH 4.5 the 2′-phosphate becomes protonated (pK = 6.5) carrying a single negative charge. Glu256 is likely to protonate, allowing a favourable hydrogen bond to the 2′-phosphate group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Changes in protonation states tune the binding mode of 2′P-ADP-ribose. (a) At pH 7.7 both the 2′-phosphate group of the ligand and Glu256 will be negatively charged, carrying a −2 and a −1 charge, respectively. Their interaction would be highly unfavourable. The ligand binds in the same orientation observed for NADP+, anchoring the 2′-phosphate close to Arg31 and hydrogen bonding to Glu256 via the terminal ribose. (b) Binding mode observed in the KPR–2′P-ADP-ribose crystal structure reported here. At pH 4.5 the 2′-phosphate becomes protonated (pK = 6.5) carrying a single negative charge. Glu256 is likely to protonate, allowing a favourable hydrogen bond to the 2′-phosphate group.
Mentions: We interpret these data as showing that the binding mode of 2′P-ADP-ribose is reversed at pH 4.5, presumably as a consequence of changes in protonation states (Fig. 3 ▶). The second pK of the 2′-phosphate group will be around 6.4 (Mas & Colman, 1984 ▶). At pH 7.7 the 2′-phosphate will be doubly charged and will interact strongly with Arg31, whereas interaction with the carboxylate side chain of Glu256, which is also negatively charged, would be unfavourable (Fig. 3 ▶a). In contrast, at pH 4.5 the 2′-phosphate would be present as the monoanion and Glu256 will be partially protonated. Interaction with the 2′-phosphate of the ligand will then be more favourable (Fig. 3 ▶b) and is observed in the crystal structure at a hydrogen-bonding distance of 2.4 Å. These changes in protonation states have triggered an otherwise unfavourable contact between a carboxylate and phosphate group and contributed significantly to the reversed binding mode observed in the crystal.

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