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 σ70 subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and co-activating 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-σ70 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 σ70, 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

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

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 Ca2+-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

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(LN₄^PhCl)­(NO)] (<b>1</b>) does not react with established HNO targets such as Fe^III hemes or Ph₃P. However, in the presence of stoichiometric H⁺ <b>1</b> behaves as an HNO donor. Complex <b>1</b> 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 (<b>3</b>) is the end product. Complex <b>1</b> also reacts with O₂ to yield the corresponding Co^III-η¹-ONO₂ (<b>2</b>) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}⁸ with biotargets such as Fe^III-porphyrins

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(LN₄^PhCl)­(NO)] (<b>1</b>) does not react with established HNO targets such as Fe^III hemes or Ph₃P. However, in the presence of stoichiometric H⁺ <b>1</b> behaves as an HNO donor. Complex <b>1</b> 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 (<b>3</b>) is the end product. Complex <b>1</b> also reacts with O₂ to yield the corresponding Co^III-η¹-ONO₂ (<b>2</b>) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}⁸ with biotargets such as Fe^III-porphyrins