MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Inhibition of p53 Sulfoconjugation Prevents Oxidative Hepatotoxicity and Acute Liver Failure.

BACKGROUND & AIMS: Sulfoconjugation of small molecules or protein peptides is a key mechanism to ensure biochemical and functional homeostasis in mammals. The PAPS synthase 2 (PAPSS2) is the primary enzyme to synthesize the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), in which oxidative stress is a key pathogenic event, whereas sulfation of APAP contributes to its detoxification. The goal of this study was to determine whether and how PAPSS2 plays a role in APAP-induced ALF. METHODS: Gene expression was analyzed in APAP-induced ALF in patients and mice. Liver-specific Papss2-knockout mice using Alb-Cre (Papss2ΔHC) or AAV8-TBG-Cre (Papss2iΔHC) were created and subjected to APAP-induced ALF. Primary human and mouse hepatocytes were used for in vitro mechanistic analysis. RESULTS: The hepatic expression of PAPSS2 was decreased in APAP-induced ALF in patients and mice. Surprisingly, Papss2ΔHC mice were protected from APAP-induced hepatotoxicity despite having a decreased APAP sulfation, which was accompanied by increased hepatic antioxidative capacity through the activation of the p53-p2-Nrf2 axis. Treatment with a sulfation inhibitor also ameliorated APAP-induced hepatotoxicity. Gene knockdown experiments showed that the hepatoprotective effect of Papss2ΔHC was Nrf2, p53, and p21 dependent. Mechanistically, we identified p53 as a novel substrate of sulfation. Papss2 ablation led to p53 protein accumulation by preventing p53 sulfation, which disrupts p53-MDM2 interaction and p53 ubiquitination and increases p53 protein stability. CONCLUSIONS: We have uncovered a previously unrecognized and p53-mediated role of PAPSS2 in controlling oxidative response. Inhibition of p53 sulfation may be explored for the clinical management of APAP overdose.

Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling.

Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP7 and 1,5-IP8 Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.

Sulfation of glycosaminoglycans depends on the catalytic activity of lithium-inhibited phosphatase BPNT2 in vitro.

Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent, lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 catalyzes the breakdown of 3'-phosphoadenosine-5'-phosphate, a by-product of glycosaminoglycan (GAG) sulfation. KO of BPNT2 in mice leads to skeletal abnormalities because of impaired GAG sulfation, especially chondroitin-4-sulfation, which is critical for proper extracellular matrix development. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which the loss of BPNT2 impairs sulfation remains unclear. Here, we used mouse embryonic fibroblasts (MEFs) to test the hypothesis that the catalytic activity of BPNT2 is required for GAG sulfation in vitro. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAGs, including decreased chondroitin-4-sulfate, present in Bpnt2-KO MEFs. We also demonstrate that missense mutations in Bpnt2 adjacent to the catalytic site, which are known to cause chondrodysplasia in humans, recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium (a common psychotropic medication) inhibits GAG sulfation and that this effect depends on the presence of BPNT2. Taken together, this work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology.

A structural basis for lithium and substrate binding of an inositide phosphatase.

Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis.

BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

Modulation of intestinal sulfur assimilation metabolism regulates iron homeostasis.

Sulfur assimilation is an evolutionarily conserved pathway that plays an essential role in cellular and metabolic processes, including sulfation, amino acid biosynthesis, and organismal development. We report that loss of a key enzymatic component of the pathway, bisphosphate 3'-nucleotidase (Bpnt1), in mice, both whole animal and intestine-specific, leads to iron-deficiency anemia. Analysis of mutant enterocytes demonstrates that modulation of their substrate 3'-phosphoadenosine 5'-phosphate (PAP) influences levels of key iron homeostasis factors involved in dietary iron reduction, import and transport, that in part mimic those reported for the loss of hypoxic-induced transcription factor, HIF-2α. Our studies define a genetic basis for iron-deficiency anemia, a molecular approach for rescuing loss of nucleotidase function, and an unanticipated link between nucleotide hydrolysis in the sulfur assimilation pathway and iron homeostasis.

Structural analyses of Candida albicans sterol 14α-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis.

With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as Candida albicans has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of C. albicans CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: (R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of C. albicans CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against Candida krusei and Candida glabrata, pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.

Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila.

Inositol phosphate kinase 2 (Ipk2), also known as IP multikinase IPMK, is an evolutionarily conserved protein that initiates production of inositol phosphate intracellular messengers (IPs), which are critical for regulating nuclear and cytoplasmic processes. Here we report that Ipk2 kinase activity is required for the development of the adult fruit fly epidermis. Ipk2 mutants show impaired development of their imaginal discs, the primordial tissues that form the adult epidermis. Although disk tissue seems to specify normally during early embryogenesis, loss of Ipk2 activity results in increased apoptosis and impairment of proliferation during larval and pupal development. The proliferation defect is in part attributed to a reduction in JAK/STAT signaling, possibly by controlling production or secretion of the pathway's activating ligand, Unpaired. Constitutive activation of the JAK/STAT pathway downstream of Unpaired partially rescues the disk growth defects in Ipk2 mutants. Thus, IP production is essential for proliferation of the imaginal discs, in part, by regulating JAK/STAT signaling. Our work demonstrates an essential role for Ipk2 in producing inositide messengers required for imaginal disk tissue maturation and subsequent formation of adult body structures and provides molecular insights to signaling pathways involved in tissue growth and stability during development.

Human genome-wide RNAi screen identifies an essential role for inositol pyrophosphates in Type-I interferon response.

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

Blast Injuries: From Improvised Explosive Device Blasts to the Boston Marathon Bombing.

Although most trauma centers have experience with the imaging and management of gunshot wounds, in most regions blast wounds such as the ones encountered in terrorist attacks with the use of improvised explosive devices (IEDs) are infrequently encountered outside the battlefield. As global terrorism becomes a greater concern, it is important that radiologists, particularly those working in urban trauma centers, be aware of the mechanisms of injury and the spectrum of primary, secondary, tertiary, and quaternary blast injury patterns. Primary blast injuries are caused by barotrauma from the initial increased pressure of the explosive detonation and the rarefaction of the atmosphere immediately afterward. Secondary blast injuries are caused by debris carried by the blast wind and most often result in penetrating trauma from small shrapnel. Tertiary blast injuries are caused by the physical displacement of the victim and the wide variety of blunt or penetrating trauma sustained as a result of the patient impacting immovable objects such as surrounding cars, walls, or fences. Quaternary blast injuries include all other injuries, such as burns, crush injuries, and inhalational injuries. Radiography is considered the initial imaging modality for assessment of shrapnel and fractures. Computed tomography is the optimal test to assess penetrating chest, abdominal, and head trauma. The mechanism of blast injuries and the imaging experience of the victims of the Boston Marathon bombing are detailed, as well as musculoskeletal, neurologic, gastrointestinal, and pulmonary injury patterns from blast injuries.

Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis.

Nucleotide hydrolysis is essential for many aspects of cellular function. In the case of 3',5'-bisphosphorylated nucleotides, mammals possess two related 3'-nucleotidases, Golgi-resident 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase (gPAPP) and Bisphosphate 3'-nucleotidase 1 (Bpnt1). gPAPP and Bpnt1 localize to distinct subcellular compartments and are members of a conserved family of metal-dependent lithium-sensitive enzymes. Although recent studies have demonstrated the importance of gPAPP for proper skeletal development in mice and humans, the role of Bpnt1 in mammals remains largely unknown. Here we report that mice deficient for Bpnt1 do not exhibit skeletal defects but instead develop severe liver pathologies, including hypoproteinemia, hepatocellular damage, and in severe cases, frank whole-body edema and death. Accompanying these phenotypes, we observed tissue-specific elevations of the substrate PAP, up to 50-fold in liver, repressed translation, and aberrant nucleolar architecture. Remarkably, the phenotypes of the Bpnt1 knockout are rescued by generating a double mutant mouse deficient for both PAP synthesis and hydrolysis, consistent with a mechanism in which PAP accumulation is toxic to tissue function independent of sulfation. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of sulfur assimilation and cytoplasmic PAP hydrolysis to normal liver function.

Structural basis of lipid binding for the membrane-embedded tetraacyldisaccharide-1-phosphate 4'-kinase LpxK.

The membrane-bound tetraacyldisaccharide-1-phosphate 4'-kinase, LpxK, catalyzes the sixth step of the lipid A (Raetz) biosynthetic pathway and is a viable antibiotic target against emerging Gram-negative pathogens. We report the crystal structure of lipid IVA, the LpxK product, bound to the enzyme, providing a rare glimpse into interfacial catalysis and the surface scanning strategy by which many poorly understood lipid modification enzymes operate. Unlike the few previously structurally characterized proteins that bind lipid A or its precursors, LpxK binds almost exclusively to the glucosamine/phosphate moieties of the lipid molecule. Steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and characterization of N-terminal helix truncation mutants uncovers the role of this substructure as a hydrophobic membrane anchor. These studies make critical contributions to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates and provide a structural template for designing small molecule inhibitors targeting this essential kinase.

Inositol pyrophosphates modulate S phase progression after pheromone-induced arrest in Saccharomyces cerevisiae.

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.

Multiple inositol phosphate species enhance stability of active mTOR.

Mechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP6) as shown in structures of mTOR, however it remains unclear if IP6, or any other inositol phosphate species, can activate mTOR kinase activity. Here, we show that multiple, exogenously added inositol phosphate species (IP6, IP5, IP4 and IP3) can all enhance the ability of mTOR and mTORC1 to auto-phosphorylate and incorporate radiolabeled phosphate into peptide substrates in in vitro kinase reactions. Although IP6 did not affect the apparent KM of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize an active form of mTORC1 in vitro. The effects of IP6 on mTOR were reversible, suggesting IP6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6 could alter mTOR solubility and electrophoretic mobility in SDS-PAGE in the presence of manganese, suggesting divalent cations may play a role in inositol phosphate regulation of mTOR. Together, these data suggest for the first time that multiple inositol phosphate species (IP4, IP5 and IP6) can dynamically regulate mTOR and mTORC1 by promoting a stable, active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR are well justified.

Coordinated inositide lipid-phosphatase activities of synaptojanin modulates actin cytoskeleton organization.

Synaptojanin proteins are evolutionarily conserved regulators of vesicle transport and membrane homeostasis. Disruption of synaptojanin function has been implicated in a wide range of neurological disorders. Synaptojanins act as dual-functional lipid phosphatases capable of hydrolyzing a variety of phosphoinositides (PIPs) through autonomous SAC1-like PIP 4-phosphatase and PIP2 5-phosphatase domains. The rarity of an evolutionary configuration of tethering two distinct enzyme activities in a single protein prompted us to investigate their individual and combined roles in budding yeast. Both PIP and PIP2 phosphatase activities are encoded by multiple gene products and are independently essential for cell viability. In contrast, we observed that the synaptojanin proteins utilized both lipid-phosphatase activities to properly coordinate polarized distribution of actin during the cell cycle. Expression of each activity untethered (in trans) failed to properly reconstitute the basal actin regulatory activity; whereas tethering (in cis), even through synthetic linkers, was sufficient to complement these defects. Studies of chimeric proteins harboring PIP and PIP2 phosphatase domains from a variety of gene products indicate synaptojanin proteins have highly specialized activities and that the length of the linker between the lipid-phosphatase domains is critical for actin regulatory activity. Our data are consistent with synaptojanin possessing a strict requirement for both two-domain configuration for some but not all functions and provide mechanistic insights into a coordinated role of tethering distinct lipid-phosphatase activities.

Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.

The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop-containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a precatalytic binding state, the ATP analogue AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg(2+)-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure.

Structural studies and protein engineering of inositol phosphate multikinase.

Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP(3) to IP(5), as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP(3) 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P(4) are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.