Elucidation of the phosphate binding mode of DING proteins revealed by subangstrom X-ray crystallography

Dorothee Liebschner, Mikael Elias, Sèbastien Moniot, Bertrand Fournier, Ken Scott, Christian Jelsch, Benoit Guillot, Claude Lecomte, Eric Chabrière

Research output: Contribution to journalArticlepeer-review

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Abstract

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 Å) at two different pH's (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 Å low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO42- form of phosphate.

Original languageEnglish (US)
Pages (from-to)7879-7886
Number of pages8
JournalJournal of the American Chemical Society
Volume131
Issue number22
DOIs
StatePublished - Jun 10 2009

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