title: Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family.
abstract: The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3',4,4',5,5')P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3',4,4',5,5')P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B-BiPh(3,3',4,4',5,5')P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed "moving metal" mechanism.
authors: Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath , Bath BA2 7AY, U.K.
authors: Stephen J
authors: SJ
authors: Mills
authors: Structural Genomics Consortium, Karolinska Institutet , 17177 Stockholm, Sweden.
authors: Camilla
authors: C
authors: Silvander
authors: Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath , Bath BA2 7AY, U.K.
authors: Gyles
authors: G
authors: Cozier
authors: Structural Genomics Consortium, Karolinska Institutet , 17177 Stockholm, Sweden.|Division of Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , 17177 Stockholm, Sweden.
authors: Lionel
authors: L
authors: Trésaugues
authors: Structural Genomics Consortium, Karolinska Institutet , 17177 Stockholm, Sweden.|Division of Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , 17177 Stockholm, Sweden.|School of Biological Sciences, Nanyang Technological University , 637551 Singapore.
authors: Pär
authors: P
authors: Nordlund
authors: Department of Pharmacology, University of Oxford , Mansfield Road, Oxford OX1 3QT, U.K.|Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath , Bath BA2 7AY, U.K.
authors: Barry V L
authors: BV
authors: Potter
mesh_terms: D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray; D007295:Inositol Phosphates / Q000737:chemistry* / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D000099182:Phosphoinositide 5-Phosphatases
chemical_list: D007295:Inositol Phosphates; D010744:Phosphoric Monoester Hydrolases; D000099182:Phosphoinositide 5-Phosphatases