Conformational changes in inositol 1,3,4,5,6-pentakisphosphate 2-kinase upon substrate binding: role of N-terminal lobe and enantiomeric substrate preference.
ABSTRACT: Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) catalyzes the synthesis of inositol 1,2,3,4,5,6-hexakisphosphate from ATP and IP(5). Inositol 1,2,3,4,5,6-hexakisphosphate is implicated in crucial processes such as mRNA export, DNA editing, and phosphorus storage in plants. We previously solved the first structure of an IP(5) 2-K, which shed light on aspects of substrate recognition. However, failure of IP(5) 2-K to crystallize in the absence of inositide prompted us to study putative conformational changes upon substrate binding. We have made mutations to residues on a region of the protein that produces a clasp over the active site. A W129A mutant allowed us to capture IP(5) 2-K in its different conformations by crystallography. Thus, the IP(5) 2-K apo-form structure displays an open conformation, whereas the nucleotide-bound form shows a half-closed conformation, in contrast to the inositide-bound form obtained previously in a closed conformation. Both nucleotide and inositide binding produce large conformational changes that can be understood as two rigid domain movements, although local changes were also observed. Changes in intrinsic fluorescence upon nucleotide and inositide binding are in agreement with the crystallographic findings. Our work suggests that the clasp might be involved in enzyme kinetics, with the N-terminal lobe being essential for inositide binding and subsequent conformational changes. We also show how IP(5) 2-K discriminates between inositol 1,3,4,5-tetrakisphosphate and 3,4,5,6-tetrakisphosphate enantiomers and that substrate preference can be manipulated by Arg(130) mutation. Altogether, these results provide a framework for rational design of specific inhibitors with potential applications as biological tools for in vivo studies, which could assist in the identification of novel roles for IP(5) 2-K in mammals.
Project description:Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.
Project description:myo-Inositol phosphates (IPs) are important bioactive molecules that have multiple activities within eukaryotic cells, including well-known roles as second messengers and cofactors that help regulate diverse biochemical processes such as transcription and hormone receptor activity. Despite the typical absence of IPs in prokaryotes, many of these organisms express IPases (or phytases) that dephosphorylate IPs. Functionally, these enzymes participate in phosphate-scavenging pathways and in plant pathogenesis. Here, we determined the X-ray crystallographic structures of two catalytically inactive mutants of protein-tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs) from the non-pathogenic bacteria Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) in complex with the known eukaryotic second messengers Ins(1,3,4,5)P4 and Ins(1,4,5)P3 Both enzymes bound these less-phosphorylated IPs in a catalytically competent manner, suggesting that IP hydrolysis has a role in plant pathogenesis. The less-phosphorylated IP binding differed in both the myo-inositol ring position and orientation when compared with a previously determined complex structure in the presence of myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6 or phytate). Further, we have demonstrated that PhyAsr and PhyAmm have different specificities for Ins(1,2,4,5,6)P5, have identified structural features that account for this difference, and have shown that the absence of these features results in a broad specificity toward Ins(1,2,4,5,6)P5 These features are main-chain conformational differences in loops adjacent to the active site that include the extended loop prior to the penultimate helix, the extended ?-loop, and a ?-hairpin turn of the Phy-specific domain.
Project description:Inositol-1,4,5-trisphosphate (IP<sub>3</sub>) is an important second messenger and one of the products of phosphoinositide-specific phospholipase C (PIPLC)-mediated phosphatidylinositol (4,5) bisphosphate (PIP<sub>2</sub>) hydrolysis. However, the function of IP<sub>3</sub> in cotton is unknown. Here, we characterized the function of <i>GhPIPLC2D</i> in cotton fiber elongation. <i>GhPIPLC2D</i> was preferentially expressed in elongating fibers. Suppression of <i>GhPIPLC2D</i> transcripts resulted in shorter fibers and decreased IP<sub>3</sub> accumulation and ethylene biosynthesis. Exogenous application of linolenic acid (C18:3) and phosphatidylinositol (PI), the precursor of IP<sub>3</sub>, improved IP<sub>3</sub> and myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP<sub>6</sub>) accumulation, as well as ethylene biosynthesis. Moreover, fiber length in <i>GhPIPLC2D</i>-silenced plant was reduced after exogenous application of IP<sub>6</sub> and ethylene. These results indicate that <i>GhPIPLC2D</i> positively regulates fiber elongation and IP<sub>3</sub> promotes fiber elongation by enhancing ethylene biosynthesis. Our study broadens our understanding of the function of IP<sub>3</sub> in cotton fiber elongation and highlights the possibility of cultivating better cotton varieties by manipulating <i>GhPIPLC2D</i> in the future.
Project description:The c.d. spectrum of oxyhaemoglobin from Camelus dromedarius is significantly affected by the presence of inositol hexakisphosphate. Correlation with O2-binding measurements shows that these dichroic changes parallel the functional properties of the protein. The optical modifications suggest that, in contrast with human haemoglobin, the conformational changes induced by inositol hexakisphosphate on dromedary oxyhaemoglobin are mainly attributable to a local change of the tertiary structure reminiscent of that of the deoxy derivative, the quaternary conformation seeming to be almost unaffected. The results provide direct evidence of the existence on the protein of two distinct sites for polyanions.
Project description:Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.
Project description:We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.
Project description:The role of chloride ions in modulating polyanion-induced conformational changes in haemoglobin from the dromedary (Camelus dromedarius) has been investigated. The results obtained have shown that: in the ferric derivative at pH 6.5 the effect of single polyanion (dextran sulphate and inositol hexakisphosphate) on the conformation is essentially local, thus involving only the tertiary structure of the protein; the presence of chloride ions at a concentration close to the physiological value (i.e. 150 mM) is essential to induce quaternary conformational changes in the polyanion-ferric protein system; comparison between structural and functional data correlates polyanion-induced tertiary conformational changes with changes in the value of midpoint potential, E'0, and quaternary changes with co-operativity.
Project description:Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.
Project description:The effect of inositol hexakisphosphate on the redox equilibria and on the c.d. spectra of ferric derivatives of haemoglobin from Camelus dromedarius has shown that: two distinct functionally relevant binding sites for polyanions are present on the protein; conformational changes promoted by inositol hexakisphosphate are largely dependent on spin state of the iron; tertiary and quaternary changes are not necessarily linked; structures induced by polyanions can be mixed forms that are neither T-state nor R-state.
Project description:Phytate (inositol hexakisphosphate, IP6) is a regulator of intracellular signaling, a highly abundant animal antinutrient, and a phosphate store in plant seeds. Here, we report a requirement for inositol polyphosphate kinases, AtIPK1 and AtIPK2beta, for the later steps of phytate synthesis in Arabidopsis thaliana. Coincident disruption of these kinases nearly ablates seed phytate without accumulation of phytate precursors, increases seed-free phosphate by 10-fold, and has normal seed yield. Additionally, we find a requirement for inositol tetrakisphosphate (IP4)/inositol pentakisphosphate (IP5) 2-kinase activity in phosphate sensing and root hair elongation. Our results define a commercially viable strategy for the genetic engineering of phytate-free grain and provide insights into the role of inositol polyphosphate kinases in phosphate signaling biology.