Elucidation of the Phosphate Binding Mode of DING Proteins Revealed by Subangstrom X-ray Crystallography

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• 作者：Dorothee Liebschner ; Mikael Elias ; Sbastien Moniot ; Bertrand Fournier ; Ken Scott ; Christian Jelsch ; Benoit Guillot ; Claude Lecomte ; Eric Chabrire
• 刊名：Journal of the American Chemical Society
• 出版年：2009
• 出版时间：June 10, 2009
• 年：2009
• 卷：131
• 期：22
• 页码：7879-7886
• 全文大小：264K
• 年卷期：v.131,no.22(June 10, 2009)
• ISSN：1520-5126

文摘

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.