Antibiotic production and resistance pathways in Streptomyces are dictated by the interplay of transcriptional regulatory proteins that trigger downstream responses via binding to small diffusible molecules. To decipher the mode of DNA binding and the associated allosteric mechanism in the sub-class of transcription factors that are induced by γ-butyrolactones, we present the crystal structure of CprB in complex with the consensus DNA element to a resolution of 3.25 Å. Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove. The crystal structure revealed that, CprB is bound to DNA as a dimer of dimers with the mode of binding being analogous to the broad spectrum multidrug transporter protein QacR from the antibiotic resistant strain Staphylococcus aureus. It was demonstrated that the CprB displays a cooperative mode of DNA binding, following a clamp and click model. Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator. Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).
Post-translational methylation of
rRNA at select positions is a
prevalent resistance mechanism adopted by pathogens. In this work,
KsgA, a housekeeping ribosomal methyltransferase (rMtase) involved
in ribosome biogenesis, was exploited as a model system to delineate
the specific targeting determinants that impart substrate specificity
to rMtases. With a combination of evolutionary and structure-guided
approaches, a set of chimeras were created that altered the targeting
specificity of KsgA such that it acted similarly to erythromycin-resistant
methyltransferases (Erms), rMtases found in multidrug-resistant pathogens.
The results revealed that specific loop embellishments on the basic
Rossmann fold are key determinants in the selection of the cognate
RNA. Moreover, in vivo studies confirmed that chimeric constructs
are competent in imparting macrolide resistance. This work explores
the factors that govern the emergence of resistance and paves the
way for the design of specific inhibitors useful in reversing antibiotic
resistance.
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