The antibiotic sorangicin A inhibits promoter DNA unwinding in a Mycobacterium tuberculosis rifampicin-resistant RNA polymerase

Abstract

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (Rif^R) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of Rif^R RNAPs, including the most prevalent clinical Rif^R RNAP substitution found in Mtb infected patients (S456>L of the β subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the Rif^R S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

Keywords: RNA polymerase; antibiotics; cryo-electron microscopy; multidrug-resistant Mycobacterium tuberculosis; sorangicin A.

Fig. 1.

Sor and Rif inhibition of WT and S>L mycobacterial RNAPs. (A) Schematic of the steps of bacterial transcription initiation. The cartoon shows steps of bacterial transcription initiation, starting from RP1 to the elongation complex. The core RNAP is a pink circle, the promoter DNA is colored dark gray, and the active site Mg²⁺ is depicted as a yellow sphere. (B) Chemical structures of Rif and Sor. (C) IC₅₀ values for Rif and Sor on Mtb and Msm WT- and S>L-RNAPs calculated from two experiments (example gels in D and SI Appendix, Fig. S1). All reactions contained the transcription factor RbpA. The sequence of the promoter used for these assays is shown in SI Appendix, Fig. S1. (D) Transcriptional profile of Mtb WT (Left) and S456L (Right) RNAPs as a function of antibiotic concentrations. The full-length, 71-nucleotide run-off transcript and the abortive product (pppGpUpU*) are indicated.

Fig. 2.

The structural basis of Sor binding and inhibition of Msm Rif^R RNAP β S447L. (A) Upstream fork-promoter DNA used for crystallization of Msm RNAP/Sor structures. The −10 and −35 promoter elements are colored yellow. The extended −10 element is colored green. (B and D) 2Fo-Fc density maps (blue mesh) of Msm WT (B) or S447L (C and D) RNAPs with superimposed atomic models. The RNAP β subunit is cyan, but FL2 is dark blue. Sor (when present) is green. All structures contained the essential transcription factor RbpA. (B) Msm WT-RNAP with Sor. Amino acids defining the range of the FL2 (E462 and G450) are labeled, as are Sor-interacting amino acids discussed in the Conformational Changes in the Rif Pocket Caused by the S447L Mutation. (C) Msm S447L-RNAP with Sor, illustrating a loss of density for FL2. Notable is the loss of density for L447 and R456, whose interactions with Sor are lost. (D) Msm S447L-RNAP without Sor, illustrating a loss of density for FL2, as well as neighboring loops containing Q484 and P480. (E–H) Msm antibiotic/RNAP crystal structures (E–G) or model (H). The RNAP is shown as a molecular surface, color-coded according to the key on the left, except FL2 is dark blue. The antibiotics are shown as CPK spheres (Sor, green carbon atoms; Rif, yellow carbon atoms). The boxed area is magnified in Insets. ESAs for the antibiotics are shown at the bottom (28). (E) Msm WT-RNAP with Sor. (F) Msm WT-RNAP with Rif (PDB ID code 6CCV) (25). (G) Msm S447L-RNAP with Sor. The ΔESA is the increase in the Sor ESA for the S447L-RNAP vs. WT-RNAP. (H) Model of Msm S447L-RNAP with Rif, generated by superimposing the Rif from 6CCV onto the RNAP structure from F. The ΔESA is the increase in the Rif ESA for the S447L-RNAP model vs. WT-RNAP.

Fig. 3.

Cryo-EM experiments demonstrate Mtb RNAP S456L-RNAP/Sor can form RP2 but not RPo. (A) Duplex AP3 promoter DNA (11) used for de novo unwinding in cryo-EM structures of Mtb RNAP with Sor. (B–E) Cryo-EM structures of Mtb S456L (B and C) or WT (D and E) RNAP holoenzyme/RbpA/CarD/Sor/AP3 complexes. RNAPs and transcription factors were incubated first with Sor and then duplex AP3 promoter DNA (A). At the top of B–E, difference maps around the Sor binding site are shown as a blue mesh (normalized and contoured at 7σ). Sor is shown in stick format in green (the Sor in B is modeled to show the Sor position; Sor is not modeled in the structure). At the bottom of B–E, the cryo-EM structures are shown, with the RNAP shown as a transparent molecular surface. The RNAP active site Mg²⁺ is shown as a yellow sphere, and blue is the T-strand +1 base modeled in for reference. Difference cryo-EM density for Sor (green, normalized, and contoured at 7σ) and DNA (red) is shown. The maps are normalized to each other (PyMOL). (B) Mtb S456L-RPo class (SI Appendix, Fig. S4A); Sor difference density is fragmented and nearly absent. (C) Mtb S456L-RP2 class (SI Appendix, Fig. S4A) shows strong density for Sor. (D) Mtb WT-RPo class (SI Appendix, Fig. S4B) shows strong density for Sor. (E) Mtb WT-RP2 class (SI Appendix, Fig. S4B) shows strong density for Sor.

Fig. 4.

Positional and torsional changes of FL2 and Sor. The central core of WT-RPo/apo (PDB ID code 6EDT; yellow backbone tube in A–D) (11, 32) was used as a reference to align the Mtb WT- and S456L-RNAP cryo-EM structures with Sor to compare the positional and conformational changes of the FL2 relative to the rest of RNAP. SI Appendix, Table S3 lists the rmsd values for the superpositions. In A–D, the DNA (T-strand, dark gray; NT-strand, light gray) is shown as a molecular surface. The side chains of S/L456 and L463, which approach the +2 base in the NT-strand (NT +2), are shown. (A) The S456L substitution does not significantly affect the conformation of FL2 in RPo without Sor. DNA is from PDB ID code 6EDT (WT/apo). (B) The presence of Sor does not significantly affect the conformation of FL2 in WT-RNAP RPo or RP2 structures. DNA is from PDB ID code 6EDT. (C and D) The combination of the S456L substitution, Sor, and promoter DNA (in RP2) forces an alteration of FL2 (dark blue), causing L463 to clash with the NT +2 DNA in RPo. The ratio rmsd of S456L RP2/Sor was 2.2. This ratio rmsd indicates the FL2 of S456L RP2 with Sor has greater variability than the other structures compared to the core module. (C) Sor binding to the S>L-RNAP affects the conformation of the FL2. The FL2 of WT-RPo, WT-RPo/Sor, WT-RP2/Sor, and S456L-RP2/Sor are shown. The DNA shown is from S456L-RPo. This figure illustrates that in the presence of Sor, the position of the S456L FL2 (dark blue tube) changes such that L463 clashes with the NT +2 DNA in the RPo state. (D) The FL2 of WT-RPo, S456L-RPo, and S456L-RP2/Sor are shown. The DNA shown is from S456L RPo. The collective results shown in C and D illustrate that the combination of the S456L substitution, Sor, and promoter DNA (in RP2) combine to force FL2 into a conformation that hinders the formation of RPo. (E) Sor is repositioned with minimal torsional changes upon binding to Msm S447L-RNAP with no downstream DNA. The repositioning and torsional movements of Sor were calculated by aligning the core modules (as described above for the cryo-EM structure of the Mtb RNAPs) of Msm WT-RNAP/Sor and Msm S447L-RNAP/Sor. The centers of mass of Sor from the S447L-RNAP and WT-RNAP were then calculated using PyMOL. On the left in E, Sor in the S447L-RNAP has a change in the center of mass of 1.1 Å, compared to the WT enzyme, moving toward the disordered FL2. On the right in E, no significant torsional conformation changes observed for Sor bound to the WT and S447L (rmsd of 0.064 over 58 atoms). (F) Sor, upon binding to Mtb S456L-RNAP in the presence of promoter DNA, exhibits repositioning and torsional movements. L456 atoms are shown as spheres. The repositioning and torsional movements of Sor were calculated using similar alignments of the core modules of the cryo-EM structure of the Mtb RNAPs with Sor as described in A and C, using WT RPo/Sor as the reference molecule. The center of mass (calculated using PyMOL) of Sor in the WT-RP2 structure showed a repositioning of 0.2 Å, which increased to 0.7 Å in the S456L-RNAP (arrow). Aligning the Sor molecule using the “pair fit” command in PyMOL showed that the torsional differences of Sor between the WT-RPo/Sor and WT-RP2/Sor to be 0.438 Å (over 58 atoms). The torsional differences between WT-RPo/Sor and WT-RP2/Sor to S456L RP2/Sor were 0.790 and 0.822 Å, respectively. The change in position of C30 is 2.4 Å, attributed to the S456L mutation (shown in spheres).

Fig. 5.

Model of Sor’s effect on the transcription of WT- and S>L-RNAPs. The effects of Sor and the S456L mutation on the transcription initiation steps to elongation of Mtb RNAP are shown in cartoon with the key below. (Top) WT-RNAP (and the S>L-RNAP; not shown) without Sor can proceed to the productive complex RPec. (Middle) The WT enzyme with Sor can bind double-stranded DNA (RP1) and proceed to RPitc, but Sor blocked the transition from RPitc to RPec (red X). (Bottom) The S>L-RNAP is inhibited at the transition from RP2 to RPo (red dashed X) because of the clash between the FL2 and the NT-strand DNA. The dashed X indicates that other explanations, which require additional investigations, may apply to why RPo is inhibited.