Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels.

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this "gating pore" when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open-closed gating, but strikingly, at negative voltages where "normal" gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 µM Zn2+ Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier.

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.

Hv 1 Proton Channels in Dinoflagellates: Not Just for Bioluminescence?

Bioluminescence in dinoflagellates is controlled by HV 1 proton channels. Database searches of dinoflagellate transcriptomes and genomes yielded hits with sequence features diagnostic of all confirmed HV 1, and show that HV 1 is widely distributed in the dinoflagellate phylogeny including the basal species Oxyrrhis marina. Multiple sequence alignments followed by phylogenetic analysis revealed three major subfamilies of HV 1 that do not correlate with presence of theca, autotrophy, geographic location, or bioluminescence. These data suggest that most dinoflagellates express a HV 1 which has a function separate from bioluminescence. Sequence evidence also suggests that dinoflagellates can contain more than one HV 1 gene.

Fin-fold development in paddlefish and catshark and implications for the evolution of the autopod.

The evolutionary origin of the autopod involved a loss of the fin-fold and associated dermal skeleton with a concomitant elaboration of the distal endoskeleton to form a wrist and digits. Developmental studies, primarily from teleosts and amniotes, suggest a model for appendage evolution in which a delay in the AER-to-fin-fold conversion fuelled endoskeletal expansion by prolonging the function of AER-mediated regulatory networks. Here, we characterize aspects of paired fin development in the paddlefish Polyodon spathula (a non-teleost actinopterygian) and catshark Scyliorhinus canicula (chondrichthyan) to explore aspects of this model in a broader phylogenetic context. Our data demonstrate that in basal gnathostomes, the autopod marker HoxA13 co-localizes with the dermoskeleton component And1 to mark the position of the fin-fold, supporting recent work demonstrating a role for HoxA13 in zebrafish fin ray development. Additionally, we show that in paddlefish, the proximal fin and fin-fold mesenchyme share a common mesodermal origin, and that components of the Shh/LIM/Gremlin/Fgf transcriptional network critical to limb bud outgrowth and patterning are expressed in the fin-fold with a profile similar to that of tetrapods. Together these data draw contrast with hypotheses of AER heterochrony and suggest that limb-specific morphologies arose through evolutionary changes in the differentiation outcome of conserved early distal patterning compartments.

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel.

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, H(V)1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, H(V)1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of H(V)1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H(V)1 requires an acidic group at the selectivity filter.

Voltage-gated proton channel in a dinoflagellate.

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH(V)1: it is proton-specific and activated by depolarization, its g(H)-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp(51)) results in loss of proton-specific conduction. Indirect evidence suggests that kH(V)1 is monomeric, unlike other proton channels. Furthermore, kH(V)1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E(H). This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain.

Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region, and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits, including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed splice form, Nox5S) inhibit full-length Nox5, consistent with formation of a dominant negative complex. Oligomerization of full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed, differentially tagged forms of Nox5 and occurred in a manner independent of calcium ion. Several approaches were used to show that the DH domain mediates oligomerization: Nox5 could be isolated as a multimer when the calcium-binding domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion of the DH domain eliminated oligomerization. Further, a chimera containing the transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase (CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in human aortic smooth muscle cells indicated molecular masses of ∼350 and ∼300 kDa, respectively, consistent with a tetramer being the functionally active unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is mediated by its dehydrogenase domain. As a result of oligomerization, the short, calcium-independent splice form, Nox5S, may function as an endogenous inhibitor of calcium-stimulated ROS generation by full-length Nox5.

Nox4 B-loop creates an interface between the transmembrane and dehydrogenase domains.

By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1-4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47(phox)/p67(phox), suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47(phox)/p67(phox), suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (K(d) = 58 +/- 12 nm). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes.

NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology.

The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91phox component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating.

Voltage-gated proton channels are strongly inhibited by Zn(2+), which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn(2+). We hypothesize that high-affinity Zn(2+) binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn(2+) effects were weaker in monomeric channels. Mutation of His(193) and His(140) in various combinations and in tandem dimers revealed that channel opening was slowed by Zn(2+) only when at least one His was present in each monomer, suggesting that in wild-type (WT) H(V)1, Zn(2+) binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn(2+) appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer-dimer interconversion occurs during activation of phagocytes appears to be ruled out.

Engineered high-affinity zinc binding site reveals gating configurations of a human proton channel.

The voltage-gated proton channel (HV1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hHV1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+ in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pHo 7 suggests additional groups contribute. We hypothesized that Asp185, which faces position 116 in our closed-state model, contributes to Zn2+ chelation. Confirming this prediction, V116H/D185N abolished Zn2+ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+ concentration. We hypothesized that slow turn-on of H+ current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hHV1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg205 near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp185 opposes position 116 in closed channels and that Arg205 moves between them when the channel opens.

Exotic properties of a voltage-gated proton channel from the snail Helisoma trivolvis.

Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance-voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).

Histidine168 is crucial for ΔpH-dependent gating of the human voltage-gated proton channel, hHV1.

We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the voltage-gated proton channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1-changing His168 to Gln168, the corresponding residue in HtHV1-compromises the pHi dependence of gating in the human channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by <20 mV/unit. Thus, His168 is critical to pHi sensing in hHV1. His168, located at the inner end of the pore on the S3 transmembrane helix, is the first residue identified in HV1 that significantly impairs pH sensing when mutated. Because pHo dependence remains intact, the selective erosion of pHi dependence supports the idea that there are distinct internal and external pH sensors. Although His168 may itself be a pHi sensor, the converse mutation, Q229H, does not normalize the pHi sensitivity of the HtHV1 channel. We hypothesize that the imidazole group of His168 interacts with nearby Phe165 or other parts of hHV1 to transduce pHi into shifts of voltage-dependent gating.

Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation.

NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.

Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates.

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1's predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings' hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon.

INSULT: a novel mutagenesis method generating high yields of closed circular mutant DNA with one primer per mutant.

INSULT, a novel method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special "ultracompetent" cells. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single-stranded copies of circular mutation-bearing plasmid DNA, addition of a "generic" primer followed by one or more polymerase reaction cycles generates double-stranded circular DNA bearing the desired mutation.

A single-stage polymerase-based protocol for the introduction of deletions and insertions without subcloning.

A single-stage polymerase-based procedure is described that allows extensive modifications of DNA. The version described here uses the QuikChange Site-Directed Mutagenesis System kit supplied by Stratagene. The original protocol is replaced by a single-stage method in which linear production of complementary strands is accomplished in separate single primer reactions. This has proved effective in introducing insertions and deletions into large gene/vector combinations without subcloning.

A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox.

p67(phox) is the paramount cytosolic regulator of the superoxide-generating Nox of phagocytes, by controlling the conformation of the catalytic component, Nox2. The initiating event of this process is a protein-protein interaction between p67(phox) and the part of Nox2 protruding into the cytosol, known as the dehydrogenase region. The aim of this study was to identify and characterize region(s) in Nox2 acting as binding site(s) for p67(phox). For this purpose, we measured the binding of recombinant p67(phox) to an array of 91 overlapping synthetic pentadecapeptides covering the length of the dehydrogenase region (residues 288-570). We found that: 1) p67(phox) binds to a site corresponding to residues 357-383, represented by a cluster of 5 peptides (Nos. 24-28); 2) maximal binding was to peptides 24 (357-371) and 28 (369-383); 3) these shared a (369)Cys-Gly-Cys(371) triad, found to be responsible for binding; 4) the Cys-Gly-Cys triad was present in Nox2 of mammals, birds, and amphibians but was absent in other Nox; 5) substituting a Nox4 or Nox1 sequence for the Nox2 sequence in peptide 24 abolished binding; 6) replacing (369)Cys by Arg in peptide 24 (mimicking a mutation in chronic granulomatous disease) abolished binding; 7) the same replacement in peptide 28 did not affect binding, indicating the existence of an additional binding site. Our results reveal an essential role for the Cys-Gly-Cys triad in Nox2 in binding p67(phox), seconded by an additional binding region, comprising residues C terminal to Cys-Gly-Cys. The 2 regions interact with distinct partner sites in p67(phox).