Elucidation of the phosphate binding mode of DING proteins revealed by subangstrom X-ray crystallography.
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 A) 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 A 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 HPO(4)(2-) form of phosphate.
Liebschner
Dorothee
D
Cristallographie Resonnance Magnetique et Modelisations, CNRS UMR 7036 Nancy-Universite, 54506 Vandoeuvre-les-Nancy, France.
Elias
Mikael
M
Moniot
Sèbastien
S
Fournier
Bertrand
B
Scott
Ken
K
Jelsch
Christian
C
Guillot
Benoit
B
Lecomte
Claude
C
Chabrière
Eric
E
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D028044:Phosphate-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
D001426:Bacterial Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates