Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species,but not redox activity,as critical elements in cytotoxicity

Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054-7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.     

Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.     

The interaction of azurin and C-terminal domain of p53 is mediated by nucleic acids

Azurin is bacterial protein, which was been reported to promote cancer cell death in vitro. The interaction of azurin and p53 is important for the cytotoxic effect of azurin towards cancer cells. In this study, it was found that nucleic acids mediated the interaction of azurin and the C-terminal domain of p53 (residues 352-393). The results provide novel insight into the interaction, and raising the possibility that the allosteric regulation of C-terminus of p53 by nucleic acids play an important role in the interaction of p53 with azurin. Meanwhile an elongated expressed product of azurin was cloned and purified, which was found to have stronger interaction with C-terminal domain of p53. Cytotoxicity studies showed that the cytotoxic effect of this elongated expressed product of azurin was stronger than wild-type azurin. The difference found in the cytotoxic effect of azurin with various sequence may provide valuable insight for finding more effective anticancer peptides.     

Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.     

Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy

p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.     

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.     

Solvent-exposed residues located in the beta-sheet modulate the stability of the tetramerization domain of p53--a structural and combinatorial approach

The role of hydrophobic amino acids in the formation of hydrophobic cores as one of the major driving forces in protein folding has been extensively studied. However, the implication of neutral solvent-exposed amino acids is less clear and available information is scarce. We have used a combinatorial approach to study the structural relevance of three solvent-exposed residues (Tyr(327), Thr(329), and Gln(331)) located in thebeta-sheet of the tetramerization domain of the tumor suppressor p53 (p53TD). A conformationally defined peptide library was designed where these three positions were randomized. The library was screened for tetramer stability. A set of p53TD mutants containing putative stabilizing or destabilizing residue combinations was synthesized for a thermodynamic characterization. Unfolding experiments showed a wide range of stabilities, with T(m) values between 27 and 83 degrees C. Wild type p53TD and some highly destabilized and stabilized mutants were further characterized. Thermodynamic and biophysical data indicated that these proteins were folded tetramers, with the same overall structure, in equilibrium with unfolded monomers. An NMR study confirmed that the main structural features of p53TD are conserved in all the mutants analyzed. The thermodynamic stability of the different p53TD mutants showed a strong correlation with parameters that favor formation and stabilization of the beta-sheet. We propose that stabilization through hydrophobic interactions of key secondary structure elements might be the underlying mechanism for the strong influence of solvent-exposed residues in the stability of p53TD.     

p53 R175H hydrophobic patch and H‐bond reorganization observed by MD simulation

Molecular dynamics simulations probe the origins of aberrant functionality of R175H p53, which normally prevent tumorigenesis. This hotspot mutation exhibits loss of its essential zinc cofactor, aggregation, and activation of gain of function promoters, characteristics contributing to the loss of normal p53 activity. This study provided molecular level insight into the reorganization of the hydrogen bonding network and the formation of a hydrophobic patch on the surface of the protein. The hydrogen bonding network globally redistributes at the expense of the stability of the β‐sandwich structure, and surface residues reorganize to expose a 250 Ų hydrophobic patch of residues covering approximately 2% of the solvent accessible surface. These changes could both stabilize the protein in the conformation exposing the patch to solvent to mediate the reported aggregation, and cause a destabilization in the area associated with DNA binding residues to affect the specificity. The development of the patch prior to loss of zinc indicates that stabilizing the patch quickly may prevent zinc loss. Considerations for rational design of small molecule therapeutics in light of the structural insight has been discussed and it suggest the positive ring around the hydrophobic patch and conserved residues may constitute a druggable site. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 176–185, 2016.     

Pseudomonas aeruginosa cytochrome C(551): probing the role of the hydrophobic patch in electron transfer

Cytochrome c(551) from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd(1) nitrite reductase. The distribution of charged residues on the surface of c(551) is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c(551) the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23-->Asp, Pro58-->Ala and Ile59-->Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd(1) nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer.     

Modeling the interaction between the N‐terminal domain of the tumor suppressor p53 and azurin

It is known that the half life of the tumor suppressor p53 can be increased by the interaction with the bacterial protein azurin, resulting in an enhanced anti‐tumoral activity. The understanding of the molecular mechanisms on the basis of this phenomenon can open the way to new anti‐cancer strategies. Some experimental works have given evidence of an interaction between p53 and azurin (AZ); however the binding regions of the proteins are still unknown. Recently, fluorescence studies have shown that p53 partakes in the binding with the bacterial protein by its N‐terminal (NT) domain. Here we have used a computational method to get insight into this interacting mode. The model that we propose for the best complex between AZ and p53 has been obtained from a rigid‐body docking, coupled with a molecular dynamics (MD) simulation, a free energy calculation, and validated by mutagenesis analysis. We have found a high degree of geometric fit between the two proteins that are kept together by several hydrophobic interactions and numerous hydrogen bonds. Interestingly, it has emerged that AZ binds essentially to the helices H_I and H_III of the p53 NT domain, which are also interacting regions for the foremost inhibitor of p53, MDM2. Copyright © 2009 John Wiley & Sons, Ltd.     

Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopy

Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour‐suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down‐regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N‐terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein–protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti‐cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd.     

19F NMR study of the leucine-specific binding protein of Escherichia coli: mutagenesis and assignment of the 5-fluorotryptophan-labeled residues

The Escherichia coli L-leucine receptor is an aqueous protein and the first component in the distinct transport pathway for hydrophobic amino acids. L-leucine binding induces a conformational change, which enables the receptor to dock to the membrane components. To investigate the ligand-induced conformational change and binding properties of this protein, we used (19)F NMR to probe the four tryptophan residues located in the two lobes of the protein. The four tryptophan residues were labeled with 5-fluorotryptophan and assigned by site-directed mutagenesis. The (19)F NMR spectra of the partially ligand free proteins show broadened peaks which sharpen when L-leucine is bound, showing that the labeled wild-type protein and mutants are functional. The titration of L-phenylalanine into the 5-fluorotryptophan labeled wild-type protein shows the presence of closed and open conformers. Urea-induced denaturation studies support the NMR results that the wild-type protein binds L-phenylalanine in a different manner to L-leucine. Our studies showed that the tryptophan to phenylalanine mutations on structural units linked to the binding pocket produce subtle changes in the environment of Trp18 located directly in the binding cleft.     

Structural, dynamical and functional aspects of the inner motions in the blue copper protein azurin

Molecular dynamics was applied to dissect out the internal motions of azurin, a copper protein performing electron transfer. Simulations of 16.5 ns were analyzed in search of coordinated displacements of amino acid residues that are important for the protein function. A region with high conformational instability was found in the 'southern' end of the molecule, far away from the copper site and the binding sites for the redox partners of azurin. By excluding the 'southern' region from the subsequent analysis, correlated motions were identified in the hydrophobic patch that surrounds the protein active site. The simulation results are in excellent agreement with recent NMR data on azurin in solution [A. V. Zhuravleva, D. M. Korzhnev, E. Kupce, A. S. Arseniev, M. Billeter, V. Y. Orekhov, Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures, J. Mol. Biol. 342 (2004) 1599-1611] and suggest a rationale for cooperative displacements of protein residues that are thought to be critical for the electron transfer process. A number of other structural and dynamic features of azurin are discussed in the context of the blue copper protein family and an explanation is proposed to account for the variability/conservation of some regions in the cupredoxins.     

Residues Y179 and H101 of a hydrophobic patch of factor VII are involved in activation by factor Xa

We studied factor Xa activation of human factor VII in hopes of identifying factor VII residues, not adjacent to the cleavage site, involved in this interaction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with multiple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X activation, and the residues of MM4 are located at a hydrophobic patch of factor VII on the opposite side of the catalytic domain from those in MM3. Only H101A, Y179A, and MM4 were activated significantly more slowly than the wild type. Results of our kinetic analyses showed that the catalytic efficiency of factor Xa for activation of factor VII was 176- and 234-fold higher than that for H101A andY179A, respectively. All the mutants with measurable activity had affinities for tissue factor similar to those of the wild type. The activated hydrophobic patch residues, except N100A, which is adjacent to one of the catalytic residues, had normal activities toward both a small peptide substrate and factor X. The rest of the activated mutants (except D102Q with no activity) had reduced activities toward the small substrate (except R147A) and factor X. We conclude that factor VII activation by factor Xa and factor VIIa's catalytic interaction with factor X involve different regions in the catalytic domain, and residues H101 and Y179, part of an aromatic hydrophobic patch, are specifically involved in factor Xa activation of factor VII.     

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.     

A spectrofluorometric study of the environment of tryptophans in bacteriorhodopsin

The emission spectrum of intact purple membranes of Halobacterium halobium has a very short wavelength position (the main maximum at 314 nm) and can be fitted by two spectral components, one of which (component A) corresponds to the fluorescence of buried tryptophan residues located in a highly hydrophobic rigid environment (like the single tryptophan residue in azurin), the other (component I) being due to the emission of buried tryptophan residues located in a rather polar environment. Treatment of bacteriorhodopsin by NaBH4, fragmentation of the membranes and thermal formation of vesicles result in a decrease in the contribution of component A, an increase in that of component I and the appearance of spectral components corresponding to the emission of surface tryptophan residues. Temperature induces at least two distinct changes of the fluorescence parameters of the protein: one change occurs from 45 to 65 degrees C. the other from 65 to 90 degrees C. The spectral changes correlate with the peaks of heat sorption caused by thermal transitions in the purple membrane structure and conformational changes in the protein structure. Alkaline denaturation of bacteriorhodopsin registered by tryptophan fluorescence begins at pH > 11.0.     

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction ³Zncyt + az(II) → Zncyt⁺ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrosatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.     

Electron relaxation and solvent accessibility of the metal site in wild-type and mutated azurins as determined from nuclear magnetic relaxation dispersion experiments

The interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different degrees of solvent accessibility are found. The low relaxivity of wild-type azurin agrees with a solvent-protected copper site in solution, the closest water being found at a distance of more than 5?Å from the copper. This low relaxivity contrasts with the relatively large relaxivity of the His46Gly and His117Gly azurin mutants, which shows clear evidence of copper-coordinated water. The data on the latter mutants are best analyzed in terms of one and two water molecules coordinated to the copper in His46Gly and His117Gly, respectively. The Met121His azurin mutant shows an intermediate behavior. The data are analyzed in terms of an increased solvent accessibility with respect to the wild-type azurin, resulting in semi-coordination of water at low pH. These different modes of coordination lead to different geometries, ranging from the trigonal type 1 site of wild-type azurin to the tetragonal type 2 copper sites of the His117Gly and His46Gly azurin mutants through a so-called type 1.5 site of the Met121His mutant. A correlation is found between the relaxation time (τ_s) of the unpaired electron of copper(II) and the geometry of the metal site: as the tetragonal character decreases the relaxation becomes significantly faster. τ_s values of ≤1?ns are found for the tetrahedrally distorted type 1 and type 1.5 sites and of 5–15?ns for the tetragonal type 2 sites.