We will target an essential transcription factor (NusA) that fulfils all criteria as an antibiotic target. NusA modulates transcription through interactions with RNA polymerase (RNAP). Our hypothesis is that inhibition of the NusA-RNAP interaction will have great therapeutic potential for development of a new class of antibiotics as it will directly interrupt the essential process of transcription.
The low resolution structure of NusA in complex with RNAP from Bacillus subtilis showed that NusA N-terminal domain (NusAN) binds to a region of RNAP called the β-flap and that NusAC is placed close to the RNA exit channel where it is able to interact with the emerging transcript [1]. In order to fully understand how NusA functions, a high-resolution structure of the complex is required that enables determination of the intermolecular interaction in detail. Mobli et al have already determined the high resolution solution structure NusAN and its binding partner and β-flap of RNAP [2]. But due to the low affinity of these individual domains in solution (Kd = 86 μM at 25 °C, measured by ITC) and the broadening of NMR peaks of both domains upon binding, we were unable to solve the structure of the complex either by NOEs-based NMR or crystallization. Here, we collected the restraints sourced from chemical shift perturbation, paramagnetic resonance enhancement (PRE) with MTSL labelling on only NusA and DEER with MTSL labelling on both NusA and β-flap. Then we will use all restraints to model the complex structure between NusA and β-flap.
We have also screened a compound library for the hits which could bind to NusAN through 1D saturation transfer difference (STD) NMR and further validation through 2D 15N-HSQC. STD revealed 30 compounds binding NusAN, six of which were confirmed to bind NusAN by 2D 15N-HSQC.