Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants - identification of 11 novel mutations in CYBB.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910 , X91- or X91+ ), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910 -CGD and two X91- -CGD). One X910 -CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91- -CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91- -CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91- -CGD patients correlates with mild clinical forms of CGD, whereas X910 -CGD and X91+ -CGD cases remain the most clinically severe forms.

Detection and quantitative analysis of two independent binding modes of a small ligand responsible for DC-SIGN clustering.

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor (CLR) present, mainly in dendritic cells (DCs), as one of the major pattern recognition receptors (PRRs). This receptor has a relevant role in viral infection processes. Recent approaches aiming to block DC-SIGN have been presented as attractive anti-HIV strategies. DC-SIGN binds mannose or fucose-containing carbohydrates from viral proteins such as the HIV envelope glycoprotein gp120. We have previously demonstrated that multivalent dendrons bearing multiple copies of glycomimetic ligands were able to inhibit DC-SIGN-dependent HIV infection in cervical explant models. Optimization of glycomimetic ligands requires detailed characterization and analysis of their binding modes because they notably influence binding affinities. In a previous study we characterized the binding mode of DC-SIGN with ligand 1, which shows a single binding mode as demonstrated by NMR and X-ray crystallography. In this work we report the binding studies of DC-SIGN with pseudotrisaccharide 2, which has a larger affinity. Their binding was analysed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol and molecular modelling. These studies demonstrate that in solution the complex cannot be explained by a single binding mode. We describe the ensemble of ligand bound modes that best fit the experimental data and explain the higher inhibition values found for ligand 2.

Crystal structure of the Rac1-RhoGDI complex involved in nadph oxidase activation.

A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.

Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox.

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).

The active N-terminal region of p67phox. Structure at 1.8 A resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease.

Upon activation, the NADPH oxidase from neutrophils produces superoxide anions in response to microbial infection. This enzymatic complex is activated by association of its cytosolic factors p67(phox), p47(phox), and the small G protein Rac with a membrane-associated flavocytochrome b(558). Here we report the crystal structure of the active N-terminal fragment of p67(phox) at 1.8 A resolution, as well as functional studies of p67(phox) mutants. This N-terminal region (residues 1-213) consists mainly of four TPR (tetratricopeptide repeat) motifs in which the C terminus folds back into a hydrophobic groove formed by the TPR domain. The structure is very similar to that of the inactive truncated form of p67(phox) bound to the small G protein Rac previously reported, but differs by the presence of a short C-terminal helix (residues 187-193) that might be part of the activation domain. All p67(phox) mutants responsible for Chronic Granulomatous Disease (CGD), a severe defect of NADPH oxidase function, are localized in the N-terminal region. We investigated two CGD mutations, G78E and A128V. Surprisingly, the A128V CGD mutant is able to fully activate the NADPH oxidase in vitro at 25 degrees C. However, this point mutation represents a temperature-sensitive defect in p67(phox) that explains its phenotype at physiological temperature.

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein.

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.

The NAD(P)H:flavin oxidoreductase from Escherichia coli. Evidence for a new mode of binding for reduced pyridine nucleotides.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2'-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.

The manganese-containing ribonucleotide reductase of Corynebacterium ammoniagenes is a class Ib enzyme.

Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mn-containing RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.

Is the NAD(P)H:flavin oxidoreductase from Escherichia coli a member of the ferredoxin-NADP+ reductase family?. Evidence for the catalytic role of serine 49 residue.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH. The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group. It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies. A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring. Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine. kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme. Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified. This suggests that serine 49 is involved in the binding of the isoalloxazine ring. Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation. This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction. In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor. Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.

Cys5 and Cys214 of NAD(P)H:flavin oxidoreductase from Escherichia coli are located in the active site.

The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH. A sequential ordered mechanism, with NADPH binding first, operates. Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group. It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-ethylmaleimide (MalNEt). Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved. Altogether this suggested a crucial role of cysteine residues for catalysis. The three cysteine residues were mutated to serine residues. Single-mutant and double-mutant enzymes were as active as the wild-type and Km values for both substrates remained the same. Cysteine residues are thus not important for activity. Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation. In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation. This strongly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH.

The mechanism and substrate specificity of the NADPH:flavin oxidoreductase from Escherichia coli.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPh or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2'-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.

Inactivation of ribonucleotide reductase by nitric oxide.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.