Conformational dynamics of C-terminal partially truncated RpoE10
The truncated SnoaL_2 domain model of the RpoE10 (Del1) was subjected to MD simulations for 200 ns trajectories and analyzed to demonstrate the experimentally observed effect of the DGGGR motif on promoter activation without NPDKV motif (Figure 1). Strikingly, the truncated SnoaL_2 domain containing the DGGG motif showed consistent and stable interactions with the Οƒ2-Οƒ4 domain across the snapshots of the 200ns simulation time (Figure 5 A, B, C; inset view). Furthermore, we observed stable conformation in the backbone RMSD of 2, 𝛔4 combined 𝛔2-𝛔4 domain, DGGGR, and WLPEP motifs throughout the simulation (Figure. 5D). In RpoE10 (Del1), after initial fluctuations in the RMSD, both the 𝛔2domain and combined 𝛔2-𝛔4 domain showed an upsurge in RMSD value at ~125 ns and then stabilized in remaining ~75 ns (Figure 5D). The RMSD analysis for 𝛔2,4 and 𝛔2-𝛔4domain along with and linker strand segments suggests that backbone conformations for RpoE10(Del1) were stable even after partial truncation of the SnoaL_2 domain. Furthermore, to ensure the stabilized and homogenous conformers, the histogram of RMSD was plotted against the number of conformers for RpoE10(Del1). In RpoE10(Mut1), the RMSD for most of the 𝛔2-𝛔4 domain conformers was restricted to ∼6-7Γ… (Figure 5E). This analysis suggests that the truncated SnoaL_2 derivative of RpoE10 containing the DGGG motif adopts stable conformations of 𝛔2-𝛔4domain.
We also evaluated the compactness (Rg value) of the individual 𝛔2,4, combined 𝛔2-𝛔4 domain, and full-length RpoE10(Del1) model. A steady Rg value for 𝛔2,4 and combined 𝛔2-𝛔4 domain of RpoE10(Del1) was obtained (Figure 5G) Furthermore, the inter-domain distance motions between 𝛔2 and 𝛔4 domains further mirrors the stable and compact 𝛔2-𝛔4domain (Figure 5H). A histogram plot of RMSD against the number of conformers for RpoE10 (Del1) showed that most of the 𝛔2-𝛔4 domain conformers were restricted to ~1.76-1.80 nm (Figure. 5F). This analysis suggests that the truncated SnoaL_2 domain containing the DGGG motif may constraint the 𝛔2-𝛔4 domains to a stable and compact protein structure required for promoter recognition and activation. Therefore, the observations that SnoaL_2 domain constraints Οƒ domain to a stable and compact structure18 can be attributed to the DGGG motifs of Snoal_2 domain.
We constructed and compared the RMSF plot of RpoE10 (Del1) with RpoE10 and RpoE10 (Mut1) to assess the impact of the DGGGR motif onto the key residue positions of the truncated SnoaL_2 domain model (Figure 5I). We focussed on key residue segments essential in initiating the promoter recognition and determining the ECF Οƒ factors’ specificity at the βˆ’10 and -35 promoter elements in ECF Οƒ-dependent promoters31,32. Intriguingly, as shown and marked in Figure 5I, these key residue positions of the RpoE10 showed contrasting fluctuations. As compared to RpoE10, an enhanced fluctuation was noticed in the L3-loop (residues 46-52) of Οƒ2 in both RpoE10(Del1) and RpoE10(Mut1). The enhanced flexibility of the L3-loop β€œspecificity loop” is known to favor the -10 promoter recognition32. We also noticed a stable peak in the residues segment 135-151 of Οƒ4 domain of RpoE10(Del1) and RpoE10(Mut1), both. This segment is known to constitute a helix-turn-helix motif that interacts with the βˆ’35 element of the promoter DNA 31(Figure 5I). Therefore, our MD simulation analysis of truncated SnoaL_2 domain, without NPDKV motif, containing the DGGG motif suggests that promoter recognition and enhanced activation is due to the stable interactions of DGGG motif with the compact conformations of 𝛔2-𝛔4domain, enhanced flexibility of the -10 recognizing L3-loop, and stability of the helix-turn-helix motif that interacts with the βˆ’35 element of the promoter DNA.