Structural characterization of Mycobacterium tuberculosis RNA polymerase binding protein A (RbpA) and its interactions with sigma factors

The RNA polymerase binding protein A (RbpA) is a 13 kDa protein, encoded by the gene Rv2050, that was shown to be essential for the growth and survival of the important human pathogen Mycobacterium tuberculosis. Although is not clear yet why RbpA is essential in M. tuberculosis, significant progress has been made in the characterization of the protein. For instance, it was shown that RbpA binds to the β-subunit of the RNA polymerase (RNAP) and activates transcription. Interestingly, it was reported that RbpA can enhance the transcription activity of the RNAP containing the primary σ-subunit σ[superscript A] but does not have any detectable effect if the RNAP is associated with the alternative σ-subunit σ[superscript F]. Moreover, it was also shown that RbpA might influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. The research project described in this thesis contributes to the ongoing efforts to characterize RbpA by providing the structure of the protein and identifying the principle σ-subunit σ[superscript A], and the principle-like σ-subunit σ[superscript B], as interaction partners. The solution structure of RbpA reveals the presence of a central structured region and highly dynamic N- and C- termini. Both termini are involved in the formation of a tight complex with the σ-subunit but only the C-terminal region appears to be essential for this interaction. The finding that RbpA also binds to the RNAP σ-subunit suggests new possibilities for the mechanism of action used by RbpA to activate transcription. Furthermore, preliminary data obtained using a ΔRv2050 conditional mutant strain of M. tuberculosis suggest that the interaction with the σ-subunit is essential for the functionality of RbpA

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of <i>in silico</i> fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers

Mycobacterium tuberculosis RNA Polymerase-binding Protein A (RbpA) and Its Interactions with Sigma Factors

RNA polymerase-binding protein A (RbpA), encoded by Rv2050, is specific to the actinomycetes, where it is highly conserved. In the pathogen Mycobacterium tuberculosis, RbpA is essential for growth and survival. RbpA binds to the β subunit of the RNA polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. Here we report the solution structure of RbpA and identify the principle sigma factor σ[superscript A] and the stress-induced σ[superscript B] as interaction partners. The protein has a central ordered domain with a conserved hydrophobic surface that may be a potential protein interaction site. The N and C termini are highly dynamic and are involved in the interaction with the sigma factors. RbpA forms a tight complex with the N-terminal domain of σB via its N- and C-terminal regions. The interaction with sigma factors may explain how RbpA stabilizes sigma subunit binding to the core RNA polymerase and thereby promotes initiation complex formation. RbpA could therefore influence the competition between principal and alternative sigma factors and hence the transcription profile of the cell