Phosphoinositide kinases play central roles in signal transduction by phosphorylating the inositol ring at specific positions. We solved the structure of the first such enzyme last year, that of type IIb phosphatidylinositol phosphate kinase. It revealed a protein kinase ATP-binding core and demonstrates that all phosphoinositide kinases belong to one superfamily. Modeling suggested that the flattened face binds to acidic phospholipids by electrostatic interactions. The enzyme binds to acidic vesicles containing phosphatidylserine, phosphatidic acid, or phosphoinositides mixed with phosphatidylcholine, but not to neutral phosphatidylcholine vesicles. Binding to acidic vesicles is abolished in the presence of 1.0 M NaCl, consistent with an essential electrostatic contribution to the free energy of binding. The +14 charge on the flattened face of the dimer was reduced to +2 in the triple mutant Lys72Glu/Lys76Glu/Lys78Glu. The mutation has no effect on dimerization, but reduces the apparent KA for 25 % phosphatidylserine: 75% phosphatidylcholine mixed vesicles by 16-fold. The reduction in binding can be accounted for by a loss of electrostatic interactions based on the finite difference solution to the Poisson-Boltzmann equation. The mutant reduces catalytic activity towards phosphatidylinositol 5-phosphate by ~50-fold. The wild-type enzyme binds half-maximally to phosphatidylinositol (4,5) bisphosphate- containing vesicles at a mole fraction of 0.3 % in a phosphatidylcholine background, as compared to 22 % mole fraction phosphatidylserine. The binding to phosphatidylinositol (4,5) bisphosphate-containing membranes is less sensitive to salt and to the triple mutation than binding to phosphatidylserine-containing membranes, suggesting that at least part of phosphatidylinositol (4,5) bisphosphates interaction with the enzyme is independent of the flattened face. We concluded that the flattened face of type IIb phosphatidylinositol phosphate kinase binds to membranes through nonspecific interactions, and that this interaction is essential for efficient catalysis.While type I and type II PIP kinases are highly related by sequence, they are functionally nonredundant, localize to different subcellular compartments and phosphorylate distinct substrates. The molecular basis of these differences in function, specificity and compartmentalization is unknown. In collaboration with Richard Anderson at the Univ. of Wisconsin, we found that a region spanning the kinase active core, termed the activation loop, determines both, enzymatic specificity and subcellular localization of PIP kinases. Thus, the activation loop may be target for spatial and temporal regulation of PIP kinase function.Phosphatidylinositol 3- phosphate regulates membrane trafficking and receptor signaling pathways by interacting with the FYVE domains of its target proteins. Here we report the crystal structure at 1.15 A resolution of the FYVE domain of the vacuolar protein sorting protein, Vps27p. The structure is built up from two antiparallel beta sheets and an a helix. It is stabilized by one 4-Cys and one 3-Cys, 1-His Zn2+-binding cluster. The core secondary structures are topologically similar to those of a rabphilin-3A Zn2+-binding domain and to the C1 and LIM domains, while two loops are unique to the FYVE domain. The phosphatidylinositol 3- phosphate binding site is located in a shallow pocket on one side of the domain formed by the characteristic (R/K)(R/K)HHCR motif on beta1, a Trp from the unique N-terminal loop, and an Arg from beta4. An Asp residue and the C-terminal carboxylate from a lattice contact occupy the phosphatidylinositol 3-phosphate binding site, showing how anionic ligands can bind to the specificity pocket. The tip of the FYVE domain has basic and hydrophobic surfaces that may interact non-specifically with phospholipid membranes. Our analysis of the structure suggests how FYVE domains target proteins to phosphatidylinositol 3-phosphate- containing membranes by a combination of specific binding to the inositol (1,3)-bisphosphate moiety and nonspecific electrostatic and hydrophobic interactions with the phospholipid bilayer. - phosphoinositide kinase, PIP kinase, protein structure, x-ray crystallography, enzyme mechanism, FYVE domain
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