X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins

Abstract

Rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the β subunit, blocking the path of the RNA transcript. The C3'-tail of benzoxazinorifamycin fits a cavity between the β subunit and σ factor. We propose that in addition to blocking RNA exit, the benzoxazinorifamycin C3'-tail changes the σ region 3.2 loop position, which influences the template DNA at the active site, thereby reducing the efficiency of transcription initiation. This study supports expansion of structure-activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential.

Figure 1

Structures of rifampin (RMP), rifalazil (bxRIF2a), and benzoxazinorifamycins (bxRIF2b–2d).

Figure 2

Electron density maps of RMP/bxRIF and molecular details of RNAP•RMP/bxRIF interactions A) 2Fo-Fc maps of RMP (left), bxRIF2b (center) and bxRIF2c (right) (cyan color meshes, sigma cutoff = 3). The C3-tail and C3′-tail are highlighted by yellow. B) Molecular details of the RNAP•RMP/bxRIF interactions. Fork loop 2 (green, residues 533–548, Arg540), positions of Rif resistant mutants (D516, H526 and S531) labeled, beta subunit residues from 1240 to the C-terminus have been removed for clarity, Sigma finger (orange, residues 513–516).

Figure 3

Model of transcription initiation complex in the presence of bxRIF2c A) Overlay between E. coli RNAP-bxRIF2c complex and T. thermophilus transcription initiation complex. B) Possible mechanism of C3′-tail dependent inhibition of transcription initiation.

Figure 4

Dose response curves for wild-type E. coli RNAP and bxRIF2b The average (n=2) of each data point was plotted as a function of log [bxRIF2b]. The individual data points were fit by nonlinear regression to determine the log (IC₅₀) values (see Supporting information), which were converted to the IC50 values shown in Table 2.