Structural basis of hypoxic gene regulation by the Rv0081 transcription factor ofMycobacterium tuberculosis

Abstract

ABSTRACTThe transcription factor Rv0081 ofM. tuberculosiscontrols the hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 3.3 Å resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. ArsR/SmtB family transcriptional repressors exert gene regulation by reversible metal binding. Hypoxia in general is sensed by bacterial transcriptional regulators via metals or Cys-mediated thiol switches. Oxygen sensing typically leads to transcriptional repressor changing its conformational state with altered DNA-binding property under different oxygen levels. Surprisingly Rv0081 neither has a metal binding domain nor does it possess Cys residues suggesting an alternate mechanism of gene regulation. Our structural analysis identified Ser 48, Ser 49, Ser 52 and Gln 53 as potential residues of Rv0081 involved in DNA binding. We probed DNA-binding of Rv0081 with electrophoretic mobility shift assay (EMSA) as well as surface plasmon resonance (SPR), where the Alanine mutants of these residues showed diminished DNA binding. Similarly, Aspartate mutants of these Ser residues was shown to fail to bind to DNA. Since, phosphorylation of various regulatory proteins is one of the important controlling mechanisms, we expected the role of Ser-phosphorylation of Rv0081 in hypoxic condition. Probing Rv0081 with anti-phosphoserine antibodies inM. tuberculosiscell lysate showed marked enhancement in the phosphorylation of Rv0081 protein under hypoxia. Overall, our structural and biochemical analysis provides the molecular basis for the regulation of Rv0081 in the latent phase of tuberculosis infection.IMPORTANCETuberculosis is one of the deadliest infectious diseases caused by the bacteriumMycobacterium tuberculosis. In about 90% of the infected people,M. tuberculosisexists in a dormant or a latent stage which can be reactivated in favorable conditions. Hypoxia (low oxygen pressure) is one of causes of dormancy. Understanding hypoxic gene regulation inM. tuberculosisis therefore an important step to understand latency. Rv0081 is a transcriptional regulator of genes expressed during hypoxia. In order to understand the mechanism by which Rv00081 regulates gene expression during hypoxia, we have solved the crystal structure of Rv0081 and identified amino acid residues which are critical in its transcriptional regulator activity. The crystal structure is suggestive of mechanism of gene regulation under hypoxia.