Ramona J. Bieber Urbauer

Solution structure and stability of the anti-sigma factor AsiA: Implications for novel functions

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli, σ70. AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix–turn–helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen/deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to σ70. The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to σ70, where an AsiA protomer is displaced from the dimer via the interaction of σ70 with the same residues in AsiA that constitute the dimer interface.

Proton-Induced Reactivity of NO– from a {CoNO}8 Complex

Research on the one-electron reduced analogue of NO, namely nitroxyl (HNO/NO–), has revealed distinguishing properties regarding its utility as a therapeutic. However, the fleeting nature of HNO requires the design of donor molecules. Metal nitrosyl (MNO) complexes could serve as potential HNO donors. The synthesis, spectroscopic/structural characterization, and HNO donor properties of a {CoNO}8 complex in a pyrrole/imine ligand frame are reported. The {CoNO}8 complex [Co(LN4PhCl)(NO)] (1) does not react with established HNO targets such as FeIII hemes or Ph3P. However, in the presence of stoichiometric H+1 behaves as an HNO donor. Complex 1 readily reacts with [Fe(TPP)Cl] or Ph3P to afford the {FeNO}7 porphyrin or Ph3P=O/Ph3P=NH, respectively. In the absence of an HNO target, the {Co(NO)2}10 dinitrosyl (3) is the end product. Complex 1 also reacts with O2 to yield the corresponding CoIII-η1-ONO2 (2) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}8 with biotargets such as FeIII-porphyrins.

Determinants of Affinity and Activity of the Anti-Sigma Factor AsiA †

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the sigma(70) subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-sigma(70) interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomer-protomer affinity in AsiA homodimers, to monomeric AsiA affinity for sigma(70), and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements were used to assess affinity and function of site-specific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer, V14, I17, and I40, resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiA-dependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation.

Localized production of human E-cadherin-derived first repeat in Escherichia coli

E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues. In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli. Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis. The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column. Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1. Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded. The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.

House fly cytochrome b5 exhibits kinetically trapped hemin and selectivity in hemin binding.

We report that cytochrome b(5) (cyt b(5)) from Musca domestica (house fly) is more thermally stable than all other microsomal (Mc) cytochromes b(5) that have been examined to date. It also exhibits a much higher barrier to equilibration of the two isomeric forms of the protein, which differ by a 180 degrees rotation about the alpha-gamma-meso axis of hemin (ferric heme). In fact, hemin is kinetically trapped in a nearly statistical 1.2:1 ratio of rotational forms in freshly expressed protein. The equilibrium ratio (5.5:1) is established only upon incubation at temperatures above 37 degrees C. House fly Mc cyt b(5) is only the second b-hemoprotein that has been shown to exhibit kinetically trapped hemin at room temperature or above, the first being cyt b(5) from the outer membrane of rat liver mitochondria (rat OM cyt b(5)). Finally, we show that the small excess of one orientational isomer over the other in freshly expressed protein results from selective binding of hemin by the apoprotein, a phenomenon that has not heretofore been established for any apocyt b(5).

Membranous adenylyl cyclase 1 activation is regulated by oxidation of N- and C-terminal methionine residues in calmodulin

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca(2+)-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress. We examined how oxidation by H2O2 of Met in CaM regulates CaM activation of AC1. We employed a series of thirteen mutant CaM proteins never assessed before in a single study, where leucine is substituted for Met, in order to analyze the effects of oxidation of specific Met. CaM activation of AC1 is regulated by oxidation of all of the C-terminal Met in CaM, and by two N-terminal Met, M36 and M51. CaM with all Met oxidized is unable to activate AC1. Activity is fully restored by the combined catalytic activities of methionine sulfoxide reductases A and B (MsrA and B), which catalyze reduction of the (S)- and (R)-MetSO stereoisomers. A small change in secondary structure is observed in wild-type CaM upon oxidation of all nine Met, but no significant secondary structure changes occur in the mutant proteins when Met residues are oxidized by H2O2, suggesting that localized polarity, flexibility and structural changes promote the functional changes accompanying oxidation. The results signify that AC1 catalytic activity can be delicately adjusted by mediating CaM activation of AC1 by reversible Met oxidation in CaM. The results are important for memory, learning and possible therapeutic routes for regulating AC1.

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor.

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

Solution structure and properties of AlgH from Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the algH gene regulates the cellular concentrations of a number of enzymes and the production of several virulence factors, and is suggested to serve a global regulatory function. The precise mechanism by which the algH gene product, the AlgH protein, functions is unknown. The same is true for AlgH family members from other bacteria. In order to lay the groundwork for understanding the physical underpinnings of AlgH function, we examined the structure and physical properties of AlgH in solution. Under reducing conditions, results of NMR, electrophoretic mobility, and sedimentation equilibrium experiments indicate AlgH is predominantly monomeric and monodisperse in solution. Under nonreducing conditions intra and intermolecular disulfide bonds form, the latter promoting AlgH oligomerization. The high‐resolution solution structure of AlgH reveals alpha/beta‐sandwich architecture fashioned from ten beta strands and seven alpha helices. Comparison with available structures of orthologues indicates conservation of overall structural topology. The region of the protein most strongly conserved structurally also shows the highest amino acid sequence conservation and, as revealed by hydrogen‐deuterium exchange studies, is also the most stable. In this region, evolutionary trace analysis identifies two clusters of amino acid residues with the highest evolutionary importance relative to all other AlgH residues. These frame a partially solvent exposed shallow hydrophobic cleft, perhaps identifying a site for intermolecular interactions. The results establish a physical foundation for understanding the structure and function of AlgH and AlgH family proteins and should be of general importance for further investigations of these and related proteins. Proteins 2015; 83:1137–1150.

Structural Studies of Calmodulin Activation of Estrogen Receptor Alpha

The goal is to determine the mechanism of calcium-dependent activation of estrogen receptor alpha (ERa) by calmodulin (CaM) and to ascertain how oxidative stress and oxidative modifications mediate the interactions between CaM, ERa and antiestrogens. Systemic endocrine/antiestrogen therapy is among the most common treatments for estrogen-dependent breast cancers. ERa is the primary target for antiestrogen therapies, and antiestrogen drugs such as tamoxifen and its metabolites (4-hydroxytamoxifen, endoxifen) bind tightly to ERa and inhibit its ability to activate transcription. Recently, it was demonstrated that CaM is an obligate ERa activator. Interestingly, antiestrogens that bind tightly to ERa also bind tightly to CaM. It has been suggested that therapeutic benefit of antiestrogens for estrogen-dependent breast cancers may derive partially from CaM antagonism. Towards our goal of understanding how CaM activates ERa, we have localized the CaM binding region of ERa and initiated structural studies to determine the structure of the complex of CaM with the ERa CaM binding region. Using NMR and SAXS we find that CaM bound to ERa is somewhat extended structurally compared to high affinity CaM complexes. Circular dichroism and fluorescence studies indicate high affinity between CaM and the CaM binding domain of ERa and that upon binding to CaM the CaM binding region of ERa adopts only partial helical character. Binding of CaM to hydrophobic antiestrogens (tamoxifen, 4-hydroxytamoxifen, endoxifen, raloxifene) is eliminated when methionine residues in CaM are oxidized. However, oxidation does not eliminate binding to ERa. Control experiments with CaM mutants (leucine for methionine) indicate methionine residues are not essential for antiestrogen binding. The results are important for understanding CaM activation of ERa and the link between oxidative stress and antiestrogen resistance development. (Supported by the DOD, the Georgia Cancer Coalition, Bruker AXS and the University of Georgia).