Rg analysis (compactness) of 𝛔-domains
The observation that NPDKV and DGGGR motifs of Snoal_2 domain significantly alter the conformations of2-𝛔4 domain was further evaluated by measuring the compactness of domains using Radius of gyration (Rg) analysis of MD simulations. A comparison between the Rg profile and average Rg value (indicated at the right margin, in Figure 3 A, B, C) of individual 𝛔2, 𝛔4, combined 𝛔2-𝛔4 domain, and full-length RpoE10, RpoE10(Mut1) and RpoE10(Mut2) indicated that the mutation in the NPDKV motif of SnoaL_2 domain constrains the 𝛔2, 𝛔4 and 𝛔2-𝛔4domains to a more compact domain structure. A reduced average Rg value of 1.71 nm for 𝛔2-𝛔4 domain of RpoE10(Mut1) as compared to both RpoE10 (1.75 nm) and RpoE10(Mut2) (1.73) was observed. In RpoE10(Mut1), although, a sharp fall in the Rg value from ~1.5 to ~1.25 after 50 ns of simulation time indicated a rise in the compactness of the 𝛔2 domain (Figure 3B), yet a steady Rg value for 𝛔2-𝛔4 and SnoaL_2 domain suggest a stable and compact protein. We also determined the minimum distance between the 𝛔2 and 𝛔4 domains as a function of simulation time using theΒ g_mindist Β utility of GROMACS (Figure 3 D, E, F). A varying degree (0.15-0.22 nm) of inter-domain distance motions between 𝛔2 and 𝛔4 domains in RpoE10 (Mut1) as compared to RpoE10 (0.15-0.17 nm) and RpoE10(Mut2) (0.15-0.17 nm) proteins suggests a favorable motion for the recognition and activation of promoter.
Furthermore, to zoom in on the differences in the compactness of2-𝛔4 domain, a histogram of RMSD was plotted against the number of conformers for RpoE10, RpoE10(Mut1), and RpoE10(Mut2). In RpoE10 (Mut1), the Rg value for 𝛔2-𝛔4 domain conformers was mainly restricted to ~1.69 nm. However, an increase in the Rg (~1.73 nm) was noticed for 𝛔2-𝛔4 conformers of both RpoE10 and RpoE10(Mut2) (Figure 3 G, H and I). The reduced Rg value across a large number of Οƒ2-Οƒ4 conformers in RpoE10(Mut1) suggests that the mutations in the NPDKV motif of SnoaL_2 domain constrain RpoE10 to a compact and stable structure favoring an enhanced activation of its promoter.