Abstract
The carbon storage regulator A (CsrA) and its homologs play an important role in coordinating the expression of bacterial virulence factors required for successful host infection. In addition, bacterial pathogens with deficiency of CsrA are typically attenuated for virulence. In 2016, the first series of small-molecule inhibitors of CsrA-RNA interaction were identified, which were found to achieve the CsrA-RNA inhibition by binding to the CsrA, without interfering with the RNA. However, the binding mechanism of these inhibitors of CsrA is not known. Herein, we applied molecular docking, molecular dynamics and binding free energy calculations to investigate the binding mode of inhibitors to CsrA. We found that the G11(RNA)-binding site is the most important binding site for CsrA inhibitors. An inhibitor with the proper size range can bind to that site and form a stable complex. We also found that inhibitors with larger size ranges bind to the entire CsrA-RNA interface, but have loose binding. However, this loose binding still resulted in inhibitory activity. The calculated binding free energy from MM/GBSA has a good correlation with the derived experimental binding energy, which might be used as a tool to further select CsrA inhibitors after a first-round of high-throughput virtual screening.
Highlights
The three-dimensional (3D) structures of carbon storage regulator A (CsrA) and its homologs from different species have been solved previously, which demonstrated highly similar structures[18,19,20,21,22,23]
RNA cartoons are shown in green. (b) The structure of one RNA bound to the edge of the RsmE dimer with the second RNA molecule omitted in the background; the binding sequence motif UCACGGAUGA is shown by the magenta line
RNA was shown as a magenta cartoon and the G10, G11, A12 of RNA core motif GGA are shown as sticks. (b) The binding pose of inhibitors of CsrA derived from Autodock
Summary
The three-dimensional (3D) structures of CsrA and its homologs from different species have been solved previously, which demonstrated highly similar structures[18,19,20,21,22,23]. When bound by CsrA, the ANGGA core folds into a loop stabilised by a 3-base pair (bp) stem of the flanking nucleotides In this clamp-like structure, the Shine-Dalgarno sequence which is part of the ribosome-binding site and marks the starting point of translation, is sequestered and translation is repressed[25,26,27]. In 2016, Hartmann et al.[31] described the discovery of the first CsrA-RNA interaction inhibitors by screening a library of small molecules They identified seven structurally diverse hits capable of inhibiting the CsrA-RNA interaction in a dose-dependent manner with an IC50 range of 4 to 106 μM. Five of the seven compounds are shown, and structures of the other two compounds were not disclosed in Hartmann’s report for intellectual property reasons As this is the first series of CsrA inhibitors to be identified, their binding mechanism is not known. We perform docking, molecular dynamics as well as free energy calculations to investigate the binding mechanism
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