Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.     

Binding of CRP to DNA-dependent RNA polymerase from E. coli: Modulation by cAMP of the interactions with free and DNA-bound holo and core enzyme

The regulatory protein CRP (or CAP) fromE. coli is shown to display two distinct patterns of binding interactions with DNA-dependent RNA polymerase. The free core enzyme, and both the core and the holo polymerase when bound to single-stranded DNA, can bind CRP in a cAMP-independent association reaction. Instead, the binding of CRP to free holoenzyme and to holo or core polymerase bound to native DNA was undetectable in the absence of cAMP. The specific ligand of CRP (cAMP) strengthens distinctively this class of interactions. In no case could any release of -factor be demonstrated. Estimates of the dissociation constants were obtained for the various binding reactions which were investigated under quasi-physiological ionic conditions. These, together with the known values of thein vivo concentrations of CRP and RNA polymerase, suggest that the interactions described may have a functional significance.     

Dynamic Fluctuations Provide the Basis of a Conformational Switch Mechanism in Apo Cyclic AMP Receptor Protein

Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP''s allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.     

The cyclic 3'',5''-adenosine monophosphate receptor protein and regulation of cyclic 3'',5''-adenosine monophosphate synthesis in Escherichia coli.

Summary Rates of synthesis of cyclic 3,5-adenosine monophosphate (cAMP) were measured in cultures of Escherichia coli aerating without a carbon source. This technique provides a representative measure of adenylate cyclase activity in the absence of inhibition caused by transport of the carbon source. Adenylate cyclase activity was found to vary more than 20-fold depending on the carbon source that had been available during growth. Synthesis of cAMP in cells aerating in the absence of the carbon source was highest when cells had been grown with glucose or fructose which inhibit adenylate cyclase activity severely. Synthesis of cAMP was much lower when cells had been grown with glycerol or succinate which cause only minimal inhibition of the activity.The variation in cAMP synthesis due to different carbon sources requires a functional cAMP receptor protein (CRP). Crp^- mutants synthesize cAMP at comparable rates regardless of the carbon source that afforded growth. A novel mutant of E. coli having a CRP no longer dependent on cAMP has been isolated and characterized. Adenylate cyclase activity in this mutant no longer responds normally to variations in the carbon source.     

Effects of glucagon and insulin on the cyclic AMP binding capacity of hepatocyte cyclic AMP-dependent protein kinase

Extracts obtained from rat hepatocytes incubated with saline, glucagon or insulin were electrophoresed on polyacrylamide gels and then assayed for cyclic (³H)AMP binding capacity. Analysis of the binding patterns demonstrated that glucagon dissociated a holoenzyme of cyclic AMP-dependent protein kinase in a dose-dependent manner. The increase in free regulatory subunits and, hence, in free catalytic subunits explains the activation of this enzyme by glucagon in the liver. Insulin decreased both the amount of cyclic (³H)AMP bound to the holoenzyme and the capacity of the enzyme to be dissociated when the extracts were incubated with increasing concentrations of this cyclic nucleotide. We propose that these insulin-induced effects are determined by an inhibition of the cyclic AMP binding capacity of this protein kinase. This mechanism could account for the inactivation of cyclic AMP-dependent protein kinase that insulin causes in the liver.Abbreviations cAMP (cyclic AMP), Adenosine 3,5 monophosphate - (³H)cAMP cyclic (³H)AMP - MIX 1-methyl-3-isobutylxanthine     

A Molecular Dynamics Study of Human Defensins HBD-1 and HNP-3 in Water

Abstract

Mammalian defensins are crucial components of the innate immune system. They are characterized by three disulfide bridges and exhibit broad spectrum antibacterial activity. The spacing between the cysteines and disulfide connectivities in the two classes of defensins, the α- and β-forms, are different. The structural motif of 3 β-strands appears to be conserved in α and β-defensins despite differences in disulfide connectivities and spacing between cysteines. In this study, Molecular Dynamics Simulations (MDS) have been carried out to study the conformational behavior of α- and β-defensins with and without disulfide bridges. Our results indicate that β-strands in the C-terminal region of HBD-1 and HNP-3 do not unfold during the course of MDS. The segment adopting α-helix in HBD-1 unfolds early during the simulations. The backbone hydrogen bonds in HBD-1 and HNP-3 are broken during MDS. When the disulfide bonds are absent, the N-terminal β-strand unfolds by 20 ns but β-strands are observed in the C-terminal region of HNP-3. HBD-1, without disulfide bridges, unfolds to a greater extent during the course of the MDS. Examination of distances between sulfur atoms of cysteines without disulfide bridges during the simulations indicate that there is no specific preference for native disulfide bridges, which could be the reason for the experimental observation of non-native disulfide bridge formation during chemical synthesis of human α- and β-defensins. Since defensins with non-native disulfide bridges are biologically active, the exact three dimensional structures observed for native HBD-1 and HNP-3 does not appear to be essential for exhibiting antibacterial activity.     

Interplay between site-specific mutations and cyclic nucleotides in modulating DNA recognition by Escherichia coli cyclic AMP receptor protein

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844-3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857-11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 microM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide approximately cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the DeltaG for CRP-DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.     

The formation of a disulfide cross-link between the two subunits demonstrates the dimeric structure of the mitochondrial oxoglutarate carrier

Isolated oxoglutarate carrier (OGC) can be cross-linked to dimers by disulfide-forming reagents such as Cu²⁺-phenanthroline and diamide. Acetone and other solvents increase the extent of Cu²⁺-phenanthroline-induced cross-linking of OGC. Cross-linked OGC re-incorporated in photeoliposomes fully retains the oxoglutarate transport activity. The amount of cross-linked OGC calculated by densitometry of scanned gels depends on the method of staining, since cross-linked OGC exhibits a higher sensitivity to Coomassie brilliant blue as compared to silver nitrate. Under optimal conditions the formation of cross-linked OGC dimer (stained with Coomassie brilliant blue) amounts to 75% of the total protein. Approximately the same cross-linking efficiency was evaluated from Western blots. Cross-linking of OGC is prevented by SH reagents and reversed by SH-reducing reagents, which shows that it is mediated by disulfide bridge(s). The formation of SS bridge(s) requires the native state of the protein, since it is suppressed by SDS and by heating. Furthermore, the extent of cross-linking is independent of OGC concentration indicating that disulfide bridge(s) must be formed between the two subunits of native dimers. The number and localization of disulfide bridge(s) in the cross-linked OGC were examined by peptide fragmentation and subsequent cleavage of disulfide bond(s) by β-mercaptoethanol. Our experimental results show that cross-linking of OGC is accomplished by a single disulfide bond between the cysteines 184 of the two subunits and suggest that these residues in the putative transmembrane helix four are fairly close to the twofold axis of the native dimer structure.     

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.Abbreviations IRS isoleucyl-tRNA synthetase - VRS valyl-tRNA synthetase - ARS arginyl-tRNA synthetase - cAMP cyclic adenosine-3,5-monophosphate - Cya adenyl cyclase Gene - CRP cAMP receptor protein - O.D. optical density     

Communications between the high-affinity cyclic nucleotide binding sites in E. coli cyclic AMP receptor protein: effect of single site mutations

The binding of adenosine 3',5'-cyclic monophosphate (cAMP) and its nonfunctional analogue, guanosine 3',5'-cyclic monophosphate (cGMP), to the adenosine 3',5'-cyclic monophosphate receptor protein (CRP) from Escherichia coli was investigated by means of fluorescence and isothermal titration calorimetry (ITC) at pH 7.8 and 25 degrees C. A biphasic fluorescence titration curve was observed, confirming the previous observation reported by this laboratory (Heyduk and Lee (1989) Biochemistry 28, 6914-6924). However, the triphasic titration curve obtained from the ITC study suggests that the cAMP binding to CRP is more complicated than the previous conclusion that CRP binds sequentially two molecules of cAMP with negative cooperativity. The binding data can best be represented by a model for two identical interactive high-affinity sites and one low-affinity binding site. Unlike cAMP, the binding of cGMP to CRP exhibits no cooperativity between the high-affinity sites. The effects of mutations on the bindings of cAMP and cGMP to CRP were also investigated. The eight CRP mutants studied were K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L. These sites are located neither in the ligand binding site nor at the subunit interface. The binding was monitored by fluorescence. Although these mutations are at a variety of locations in CRP, the basic mechanism of CRP binding to cyclic nucleotides has not been affected. Two cyclic nucleotide molecules bind to the high-affinity sites of CRP. The cooperativity of cAMP binding is affected by mutation. It ranges from negative to positive cooperativity. The binding of cGMP shows none. With the exception of the T127L mutant, the free energy change for DNA-CRP complex formation increases linearly with increasing free energy change associated with the cooperativity of cAMP binding. This linear relationship implies that the protein molecule modulates the signal in the binding of cAMP, even though the mutation is not directly involved in cAMP or DNA binding. In addition, the significant differences in the amplitude of fluorescent signal indicate that the mutations also affect the surface characteristics of CRP. These results imply that these mutations are not perturbing specific pathways of signal transmission. Instead, these results are more consistent with the concept that CRP exists as an ensemble of native states, the distribution of which can be altered by these mutations.     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.