Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation

Abstract

LicT belongs to an essential family of bacterial transcriptional antitermination proteins controlling the expression of sugar-metabolizing operons. When activated, they bind to nascent mRNAs, preventing premature arrest of transcription. The RNA binding capacity of the N-terminal domain CAT is controlled by phosphorylations of two homologous regulation modules by the phosphotransferase system (PTS). Previous studies on truncated and mutant proteins provided partial insight into the mechanism of signal transduction between the effector and regulatory modules. We report here the conformational and functional investigation on the allosteric activation of full-length LicT. Combining fluorescence anisotropy and NMR, we find a tight correlation between LicT RNA binding capacity and CAT closure upon PTS-mediated phosphorylation and phosphomimetic mutations. Our study highlights fine structural differences between activation processes. Furthermore, the NMR study of full-length proteins points to the back and forth propagation of structural restraints from the RNA binding to the distal regulatory module.

Resolving the activation mechanism of the D99N antiterminator LicT protein

“…These differences correspond to a higher and smaller aperture of dimers in CAT and PRD1 respectively (Figs. 3A-B) and are confirmed by the differences in chemical shifts observed at the respective dimer interfaces (Yang et al, 2020b) and Fig. 1D).…”

“…1B). This difference was unexpected, since by contrast and as already mentioned (Yang et al, 2020b), PRD1 resonances were remarkably well conserved between PRD1-PRD2* and LicT* (Fig. 1C).…”

“…A model of CAT-PRD1 containing the RNA binding domain and the first regulation domain from native LicT was proposed on the basis of NMR residual dipolar couplings and 15 N relaxation measurements, suggesting an opening of the CAT dimer in the inactive state (Déméné et al, 2008). We recently confirmed this hypothesis by a NMR systematic study on truncated and full-length proteins, either in their native or activated states, using 13 C-labeled methionines as markers of topology of the different dimers (Yang et al, 2020b). Although several 3D structures of LicT fragments have been reported (Fig.…”

“…S1). Their mapping on the model structure of full length activated LicT* (Yang et al, 2020b) indicates a localization at the PRD1 dimeric interface (Fig. 1D).…”

“…In a previous study, we used NMR chemical shifts of the RNA binding domain CAT to establish its structure conservation across all active LicT fragments, including CAT-PRD1*, except for marginal differences at the dimer interface (Yang et al, 2020b). We have now performed the full assignment of CAT-PRD1* resonance peaks (Yang et al, 2020a), and we can now analyze the configuration of the PRD1 module in this active state as well.…”