Figure 1 A) 𝛔2, 𝛔4, and SnoaL2 domain of RpoE10 model structure. The residues-residue interaction between WLPEP motif of 𝛔2- 𝛔4 linker, DGGGR and NPDKV motifs of SnoaL2 extension are shown in green, magenta, and cyan (ball and stick), respectively. B)Schematic representation of the deletion and mutant derivatives of RpoE10. The characteristic motifs of ECF41 type σ factor, WLPEP motif located between σ2 and σ4 regions, and other motifs (DGGGR and NPDKV) in the C-terminal extension. The first deletion derivative, RpoE10 (Del1) contained the RpoE10 sequence up to DGGGR, while the second deletion derivative RpoE10(Del2) excluded the DGGGR motif. Also, two site-directed mutants of RpoE10 were constructed: in RpoE10(Mut1) NPDKV motif was replaced with NAAAVmotif. InRpoE10(Mut2), DGGGR motif was replaced with AAAGR motif, and (C) : Effect of deletion and mutation ofDGGGR and NPDKV motifs of at the C-terminal of RpoE10 on the expression of abm:gfp fusion in A. brasilense Sp245 expressing full RpoE10, its two deletion derivatives, RpoE10(Del1) and RpoE10(Del2) and two mutant derivatives RpoE10(Mut1) and RpoE10(Mut2). Each bar represents the mean of triplicates in three independent experiments.
The analysis of 200ns MD simulation trajectories for RpoE10, RpoE10(Mut1), and RpoE(Mut2) enabled us to demonstrate the effect of mutations in the motifs 277NPDKV281and 200DGGGR204 of SnoaL_2 domain. The Root Mean Square Deviation (RMSD) was used for evaluating the stability and differences between the backbone trajectories of proteins from its initial structural conformation to its final snapshot. The smaller the RMSD, the more stable or rigid the conformation is. The backbone RMSD analysis of RpoE10, RpoE10(Mut1) and RpoE10(Mut2) system was carried out for full length, individual domains 𝛔2(1-76 amino acids), 𝛔4 (91-167 amino residues) and combined 𝛔2-𝛔4domain (1-167amino acids) (Figure 2). We observed a significant degree of conformational change in the Snoal_2 domain of RpoE10(Mut1) compared to its initial snapshot. The RpoE10 showed a stable and rigid conformation in this domain throughout the simulation. In RpoE10 (Mut1), the SnoaL_2 domain showed an upsurge in RMSD value from ~4Å to ~6Å after 20ns, as compared to RpoE10 and RpoE10 (Mut2). The RMSD value for RpoE10(Mut2) gradually increased from 4 Å to 5Å in the first 150ns and then raised from ~5 Å to 6 Å in the remaining 50ns. The RMSD analysis (with variations in RMSDs) suggested flexible yet stable backbone conformations for RpoE10(Mut1 and Mut2) as compared to the RpoE10 models (Figure 2B and C). Furthermore, to trace the impact of NPDKV and DGGGR motif on conformational dynamics of 𝛔2, 𝛔4 and SnoaL_2 domain, the RMSD profile of the NPDKV, DGGGR, and WLPEP motif of linker strand segments were also analyzed (Figure 2A, B, C). The WLPEP and NPDKV showed stable RMSD patterns comparable to each other in the case of RpoE10 and RpoE10 (Mut2). Noticeably, a mutation in the NPDKV fragment could result in a sharp rise in its RMSD profile over the 200ns trajectory and impacted the conformational dynamics of the 𝛔2, 𝛔4, and SnoaL_2 domains in RpoE(Mut1). Compared to RpoE10 and RpoE10 (Mut2), there is a significant RMSD variation in the backbone trajectory of the Snoal_2 domain, NPDKV, and DGGGR segment for RpoE10 (Mut1) indicated the influence of mutations at NPDKV over the 𝛔-domains. (Figure 2 A and C).
Additionally, to evaluate the differences in the backbone trajectory of2-𝛔4 domain, we plotted a histogram of RMSD against the number of conformers for RpoE10, RpoE10(Mut1), and RpoE10(Mut2) (Figure 2 D, E, and F). In RpoE10(Mut1), the RMSD of the 𝛔2-𝛔4 domain conformers were restricted to ∼5.8-6.5Å whereas RMSDs were heterogeneously distributed and varied from ∼4-7 Å in RpoE10 and ∼5-7 Å in RpoE10 (Mut2). This analysis suggests that the RpoE10 and RpoE10 (Mut2) forms acquire substantial conformational heterogeneity leading to an unstable system. However, a mutation in NPDKV leads to conformational stability. Therefore, the observations that SnoaL_2 domain constraints σ domain to a compact structure 18 are consistent in our RpoE10 models. The NPDKV and DGGGR motifs of the Snoal_2 domain can be attributed to significantly alter the conformations of -domains.