A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.     

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.     

Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands

The activity of the eukaryotic translation initiation factor eIF4E is modulated through conformational response to its ligands. For example, eIF4G and eIF4E-binding proteins (4E-BPs) modulate cap affinity, and thus physiological activity of eIF4E, by binding a site distal to the 7-methylguanosine cap-binding site. Further, cap binding substantially modulates eIF4E's affinity for eIF4G and the 4E-BPs. To date, only cap-bound eIF4E structures were reported. In the absence of structural information on the apo form, the molecular underpinnings of this conformational response mechanism cannot be established. We report here the first cap-free eIF4E structure. Apo-eIF4E exhibits structural differences in the cap-binding site and dorsal surface relative to cap-eIF4E. Analysis of structure and dynamics of apo-eIF4E, and changes observed upon ligand binding, reveal a molecular basis for eIF4E's conformational response to these ligands. In particular, alterations in the S4-H4 loop, distal to either the cap or eIF4G binding sites, appear key to modulating these effects. Mutation in this loop mimics these effects. Overall, our studies have important implications for the regulation of eIF4E.     

Membrane binding mechanisms of the PX domains of NADPH oxidase p40phox and p47phox

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40(phox) and p47(phox), contain PX domains. The PX domain of p40(phox) specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47(phox) has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40(phox) PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47(phox) PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40(phox) and p47(phox) proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47(phox), whereas such an interaction is absent in p40(phox).     

p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox

p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.     

Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production

The phagocyte oxidase (Phox) protein p40(phox) contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40(phox) localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS(phox) cells). Full-length p40(phox) exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40(phox) translocated to plasma membrane in a p67(phox)- and p47(phox)-dependent manner. Heterologous expression of p40(phox) markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COS(phox) cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47(phox) and p67(phox). The mutant p40(phox) (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67(phox) and p47(phox). The enhanced cytoskeleton association of p67(phox) and p47(phox) and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40(phox) interaction with p67(phox), was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40(phox); its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40(phox) with p67(phox).     

Oxidant-dependent phosphorylation of p40phox in B lymphocytes

As with the neutrophil NADPH oxidase, the B lymphocyte NADPH oxidase consists of a membrane-bound flavocytochrome b and regulatory factors including Rac and the cytosolic phox protein triad p67phox, p47phox, and p40phox. Here we demonstrate by phosphoamino acid analysis and the use of the potent PKC inhibitor GFX that, in response to stimulation of B lymphocytes with sodium orthovanadate and H(2)O(2), the p40phox component of the cytosolic phox triad is selectively phosphorylated on serine and threonine residues by a PKC-type protein kinase. The pattern of p40phox phosphorylation was closely related to the kinetics of tyrosine phosphorylation of PKC-delta, the main PKC isotype of B lymphocytes. Blocking H(2)O(2)-dependent tyrosine phosphorylation of PKC by genistein resulted in inhibition of p40phox phosphorylation. The correlation between the tyrosine phosphorylation of PKC-delta and the serine/threonine phosphorylation of p40phox, together with the inhibition of p40phox phosphorylation by rottlerin, a selective inhibitor of PKC-delta, makes the activated PKC-delta a likely candidate in the process of the oxidant-dependent phosphorylation of p40phox in B cells.     

Glycosphingolipid-facilitated membrane insertion and internalization of cobra cardiotoxin. The sulfatide.cardiotoxin complex structure in a membrane-like environment suggests a lipid-dependent cell-penetrating mechanism for membrane binding polypeptides

Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 angstroms resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.     

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.     

PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation

The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX domains of two oxidase components, p47(phox) and p40(phox), are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX domain of their p40(phox) gene, which selectively prevents binding to PtdIns3P. p40(phoxR58A/R58A) embryos do not develop normally but p40(phoxR58A/-) mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40(phox+/-) siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P-binding to p40(phox). p40(phoxR58A/-) mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.     

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

Crystal structure of yeast Esa1 suggests a unified mechanism for catalysis and substrate binding by histone acetyltransferases

Esa1 is the catalytic subunit of the NuA4 histone acetylase (HAT) complex that acetylates histone H4, and it is a member of the MYST family of HAT proteins that includes the MOZ oncoprotein and the HIV-1 Tat interacting protein Tip60. Here we report the X-ray crystal structure of the HAT domain of Esa1 bound to coenzyme A and investigate the protein's catalytic mechanism. Our data reveal that Esa1 contains a central core domain harboring a putative catalytic base, and flanking domains that are implicated in histone binding. Comparisons with the Gcn5/PCAF and Hat1 proteins suggest a unified mechanism of catalysis and histone binding by HAT proteins, whereby a structurally conserved core domain mediates catalysis, and sequence variability within a structurally related N- and C-terminal scaffold determines substrate specificity.     

Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires recruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol 3-kinase in human neutrophils

Neutrophils (polymorphonuclear leukocytes, PMNs) are vital to innate immunity and receive proinflammatory signals that activate G protein-coupled receptors (GPCRs). Because GPCRs transduce signals through clathrin-mediated endocytosis (CME), we hypothesized that platelet-activating factor (PAF), an effective chemoattractant that primes the PMN oxidase, would signal through CME, specifically via dynamin-2 activation and endosomal formation resulting in membrane translocation of cytosolic phagocyte oxidase (phox) proteins. PMNs were incubated with buffer or 2 muM PAF for 1-3 min, and in some cases activated with PMA, and O(2)(-) was measured, whole-cell lysates and subcellular fractions were prepared, or the PMNs were fixed onto slides for digital or electron microscopy. PAF caused activation of dynamin-2, resulting in endosomal formation that required PI3K and contained early endosomal Ag-1 (EEA-1) and Rab5a. The apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK signalosome assembled on Rab5a and phosphorylated EEA-1 and Rab GDP dissociation inhibitor, with the latter causing Rab5a activation. Electron microscopy demonstrated that PAF caused two distinct sites for activation of p38 MAPK. EEA-1 provided a scaffold for recruitment of the p40(phox)-p67(phox) complex and PI3K-dependent Akt1 phosphorylation of these two phox proteins. PAF induced membrane translocation of p40(phox)-p67(phox) localizing to gp91(phox), which was PI3K-, but not p47(phox)-, dependent. In conclusion, PAF transduces signals through CME, and such GPCR signaling may allow for pharmacological manipulation of these cells to decrease PMN-mediated acute organ injury.     

Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.     

Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.     

Antibody-induced dimerization of HARPTPalpha-EGFR chimera suggests a ligand dependent mechanism of regulation for RPTPalpha

We developed a system to study the function of the ectodomain of RPTPalpha, a transmembrane protein-tyrosine phosphatase, by fusing the HA-epitope tagged ectodomain of RPTPalpha to the transmembrane and intracellular domain of the epidermal growth factor receptor, EGFR, a receptor protein-tyrosine kinase that is activated by dimerization. Although the use of chemical crosslinkers shows that preformed HARPTPalpha-EGFR dimers exist, bivalent anti-HA-tag antibody activated HARPTPalpha-EGFR chimeras, suggesting this system may be used to study regulation of dimerization. We used this system to show that newborn calf serum may contain (a) potential ligand(s) for RPTPalpha. Our results suggest that RPTPalpha dimerization and thus activity may be affected by ligand binding.     

The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate.

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.     

Analysis of DBC1 and its homologs suggests a potential mechanism for regulation of Sirtuin domain deacetylases by NAD metabolites

Deleted in Breast Cancer-1 (DBC1) and its paralog CARP-1 are large multi-domain proteins, with a nuclear or perinuclear localization, and a role in promoting apoptosis upon processing by caspases. Recentstudies on human DBC1 show that it is a specific inhibitor of the sirtuin-type deacetylase, Sirt1, which deacetylates histones and p53. Using sensitive sequence profile searches and HMM-HMM comparisons weshow that the central conserved globular domain present in the DBC1 and it homologs from diverse eukaryotes is a catalytically inactive version of the Nudix hydrolase (MutT) domain. Given that Nudixdomains are known to bind nucleoside diphosphate sugars and NAD, we predict that this domain in DBC1 and its homologs binds NAD metabolites such as ADP-ribose. Hence, we propose that DBC1 and itshomologs are likely to regulate the activity of SIRT1 or related deacetylases by sensing the soluble products or substrates of the NAD-dependent deacetylation reaction. The complex domain architectures of the members of the DBC1 family, which include fusions to the RNA-binding S1-like domain, the DNA binding SAP domain and EF-hand domains, suggest that they are likely to function as integrators of distinct regulatory signals including chromatin protein modification, soluble compounds in NAD metabolism, apoptotic stimuli and RNA recognition.