Catalytic ATP-binding Site Research Articles

Light control of the peptide-loading complex synchronizes antigen translocation and MHC I trafficking

Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.

Herbimycin A abrogates nuclear factor-kappaB activation by interacting preferentially with the IkappaB kinase beta subunit.

NF (nuclear factor)-kappaB is known to be a critical transcription factor in inflammatory responses. We have reported that herbimycin A, a potent Src tyrosine kinase inhibitor, attenuates the NF-kappaB activation triggered by cytokines, bacterial endotoxin, and hydrogen peroxide. Accompanying the suppression by this agent, NF-kappaB-dependent gene expressions, such as cytokine, chemokine, and inducible-type nitric oxide, are specifically inhibited in glial cells. In the present study, we attempted to elucidate the possible target protein for herbimycin A on this pathway. We demonstrate here that herbimycin A preferentially inhibits IKK (IkappaB kinase)beta. Furthermore, substituting alanine for the cysteine at 59 (Cys59) in IKKbeta resulted in the insensitivity to herbimycin A, suggesting that this compound may interact with the Cys59 residue located near the catalytic ATP binding site. Taken together, these results indicate that herbimycin A can be considered a novel candidate for an anti-inflammatory drug agent through its specific inhibition of IKKbeta, which results in prevention of the expression of NF-kappaB-dependent genes implicated in the pathogenesis of inflammatory responses.

Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses.

A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)-associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low-molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7-tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC50 is approximately 20 micro m with a DNA substrate, but only 60 micro m with an RNA substrate. Several related analogues of TBBT were enzyme- and/or substrate-specific inhibitors. For example, 5,6-dichloro-1-(beta-d-ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC50 approximately 0.3 micro m), but much weaker with a DNA substrate (IC50 approximately 3 micro m). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 micro m. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,-tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP-binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP-binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.

Binding of Nucleotides by T4 DNA Ligase and T4 RNA Ligase: Optical Absorbance and Fluorescence Studies

The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K d between 0.1 and 20 μM. Nucleotide binding results in a decrease of absorbance at 260 nm due to π-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the π-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K d for the noncovalent site is ∼1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity.

Purealin blocks the sliding movement of sea urchin flagellar axonemes by selective inhibition of half the ATPase activity of axonemal dyneins.

Ciliary and flagellar movements are explained by active sliding between the outer doublet microtubules of an axoneme via their inner and outer dynein arms. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, blocked the motility of Triton-demembranated sea urchin sperm flagella within 5 min at concentrations above 20 microM. In a similar concentration range, purealin blocked the sliding movement of the flagellar axonemes in vitro within a few minutes judging from the turbidity measurements. The ATPase activity of axonemes was partially inhibited by purealin in a concentration-dependent manner. The maximum inhibition reached approximately 50% at concentrations above 20 microM, indicating that half the axonemal ATPase activity is sensitive to purealin. Similar results were observed on the ATPase activity of outer-arm-depleted axonemes and that of a mixture of 21S dynein and salt-extracted axonemes. On the other hand, ATPase activity of isolated 21S dynein was not inhibited by purealin. The inhibitory action of purealin on the axonemal ATPases was reversed by dilution of purealin. The effect of purealin on the double-reciprocal plot of the ATPase activity as a function of ATP concentrations showed that the inhibition was not a competitive type. In accord with this finding, purealin did not affect the vanadate-mediated UV photocleavage of axonemal dyneins. These results suggest that purealin binds reversibly to a site other than the catalytic ATP-binding site and inhibits half the ATPase activity of axonemes. Taken together, our results suggest that purealin-sensitive ATPase activity of the dynein arms plays an essential role in generating the sliding movement of flagellar axonemes.

Photochemical labeling and inhibition of Na,K-ATPase by 2-Azido-ATP. Identification of an amino acid located within the ATP binding site.

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid monolayer. In contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.

Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coli secA mutants in vivo.

The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. The secA gene from Bacillus subtilis was cloned and expressed in E. coli under the control of the lac or trc promoter. The temperature-sensitive growth and secretion defects of various E. coli secA mutants were complemented by the B. subtilis SecA protein, provided the protein was expressed at moderate levels. Under overproduction conditions, no complementation was observed. One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane. A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif. Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B. subtilis SecA protein to complement the growth and secretion defects of E. coli secA mutants. In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step. We conclude that lysine 106 is part of the catalytic ATP-binding site of the B. subtilis SecA protein, which is required for protein translocation in vivo.

PH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains.

Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.