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

ABSTRACT: 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, sigma(70). 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 sigma(70). The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to sigma(70), where an AsiA protomer is displaced from the dimer via the interaction of sigma(70) with the same residues in AsiA that constitute the dimer interface.

SUBMITTER: Urbauer JL

PROVIDER: S-EPMC122279 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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

Urbauer Jeffrey L JL Simeonov Mario F MF Urbauer Ramona J Bieber RJ Adelman Karen K Gilmore Joshua M JM Brody Edward N EN

Proceedings of the National Academy of Sciences of the United States of America 20020205 4

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, sigma(70). AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a n ...[more]

PMID: 11830637

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Project description: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.

Project description:Clostridioides (Clostridium) difficile is one of the leading causes of nosocomial diarrhea. Lysozyme is a common host defense against many pathogenic bacteria. C. difficile exhibits high levels of lysozyme resistance, which is due in part to the extracytoplasmic functioning (ECF) σ factor, σV. It has been previously demonstrated that genes regulated by σV are responsible for peptidoglycan modifications that provide C. difficile with high lysozyme resistance. σV is not unique to C. difficile however, and its role in lysozyme resistance and its mechanism of activation has been well characterized in Bacillus subtilis where the anti-σ, RsiV, sequesters σV until lysozyme directly binds to RsiV, activating σV. However, it remains unclear if the mechanism of σV activation is similar in C. difficile. Here, we investigated how activation of σV is controlled in C. difficile by lysozyme. We found that C. difficile RsiV was degraded in the presence of lysozyme. We also found that disruption of a predicted signal peptidase cleavage site blocked RsiV degradation and σV activation, indicating that the site-1 protease is likely a signal peptidase. We also identified a conserved site-2 protease, RasP, that was required for site-2 cleavage of RsiV and σV activation in response to lysozyme. Combined with previous work showing RsiV directly binds lysozyme, these data suggested that RsiV directly binds lysozyme in C. difficile, which leads to RsiV destruction via cleavage at site-1 by signal peptidase and then at site-2 by RasP, ultimately resulting in σV activation and increased resistance to lysozyme. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. We previously showed that σV and the regulon under its control were involved in lysozyme resistance. We have also shown in B. subtilis that the anti-σ RsiV acts as a direct sensor for lysozyme. which results in the destruction of RsiV and activation of σV. Here, we described the proteases required for degradation of RsiV in C. difficile in response to lysozyme. Our data indicated that the mechanism is highly conserved between B. subtilis and C. difficile.

Dataset Information

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

Publications

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

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