DISCUSSION
The characteristic C-terminal extension of the ECF41 σ was thought to act as an anti- σ factor 13. Deletion of the C-terminal domain, therefore, was assumed to activate the ECF41 σ factor. In an earlier study, the deletion of the SnoaL_2 domain of SigJ in M. tuberculosis  led to a complete loss of the ability of SigJ to activate its target promoter, indicating a positive role of the C-terminal SnoaL_2 domain of the SigJ in M. tuberculosis  18. In-silico analysis of the MtbSigJ structure also suggested that the C-terminal Snoal_2 domain may not be inhibitory 18. Based on the site-directed mutagenesis of the conserved residues of the Snoal_2 domain of RpoE10 of A. brasilense , we have shown that the DGGGR motif is located at the proximal end of the Snoal_2 domain, is required for the functionality of RpoE10.However, the NPDKV motif located at the distal end of the Snoal_2 domain is responsible for inhibiting the activity of RpoE10, indicating its negative role or anti-sigma factor-like activity.
Earlier experiments with the deletion of NPDKL and DGGGK motifs of ECF41 σ factors in B. licheniformis and R. sphaeroides have shown that NPDKL motif inhibited the activity of ECF41 σ factor, and deletion of the C-terminal Snoal_2 domain including NPDKV and DGGGK motif led to the complete loss in the ECF41 σ factor activity13. A study using Direct Coupling Analysis (DCA) identified the essential residues of ECF41Bli ofBacillus subtilis involved in the interaction between conserved residues of the flexible linker region and the conserved residues of the Snoal_2 domain. Out of the ten critical residues identified, N276 and K279 of the NPDKL were predicted to interact with the Y73 and G75 residues of the conserved consensus YVGPWLPEP motif in the linker region of the ECF41Bli . The N276A and K279A mutations at the C-terminus of ECF41Bli increased its functionality. This study clearly showed that the contact between the NPDKL and the YVGPWLPEP exerts a negative regulatory effect on the activity of ECF41 σ factor 14. One possible reason for the increased activity of RpoE10 due to the deletion of the NPDKV motif is the elimination of contact between the distal part of the C-terminal extension and the linker, which occludes binding of RpoE10 to the RNA polymerase or to the promoter. Similar to these observations, we have also shown in this study that replacing NPDKV with NAAAV in RpoE10 increased its functionality.
In ECF41Bli , G202 of the DGGGK motif was also found to interact with the Y73 of the YVGPWLPEP motif. The G202A mutation also led to an increase in the functionality of the ECF41Bli 14. However, in our study of RpoE10, we found that the replacement of DGGGR with the AAAGR led to a complete loss in its functionality. This suggests that DGGG residues in RpoE10 are required for its functionality. Because of the above, we hypothesize that the ECF41 σ factor can assume two alternative conformational possibilities: in one conformation NPDKV motif inhibits the activity of RpoE10, but in the other conformation, inhibitory interaction of NPDKV motif is prevented, leading to the activity of the ECF41 σ factor.
Here, we have demonstrated an elucidation of the role of the two conserved motifs (NPDKV and DGGGR) of the SnoaL2 domain in mediating promoter activation and elimination in RpoE10 sigma factors. A comparative Molecular Dynamics (MD) simulations, Principal Component Analysis (PCA), i.e., essential dynamics, and molecular interaction network analysis, were carried out for RpoE10 and its mutant derivatives RpoE10(Mut1), RpoE10 (Mut2), and RpoE10(Del1) for 200 ns trajectories. The structural model of Abr-RpoE10 based on the crystal structure of the Mtb-SigJ 18 provided us the opportunity to gain insight into the mechanism of promoter activation. The structural models of RpoE10were well superposed to the crystal structures, −10 promoter/ σ2 (PDB ID: 4LUP)32 and −35 promoter/ σ4 (PDB ID: 2H27) complexes 34 and adopt a conformation that can readily interact with the promoter. The central question that we addressed using MD and PCA analysis is how the NPDKV and DGGGR motifs of the SnoaL_2 domain impact the promoter activation. Essential insights into the responsible interactions at the molecular and structural level have been obtained from our MD simulation experiments. The substantial conformational changes were observed in the NPDKV and DGGGR motif in RpoE(Mut1) and RpoE10(Mut2) proteins, affecting promoter-recognition binding affinity, which is well supported by RMSD and RMSF analysis and PCA analysis. It is worth mentioning that unlike RpoE10 (Mut1) and RpoE10 (Del1), the intact NPDKV motif of wild-type RpoE10 and RpoE10(Mut2) significantly influenced the dynamic behavior of 𝛔2-𝛔4and SnoaL_2 domain. We noticed a significant disruption in the salt-bridge interaction network of highly conserved D30 of the DEAD motif of 𝛔2 domain and a conserved R76 of “Arginine tetrad73RRRR76” present near the 𝛔2linker junction. The stacking interaction between W83-R76 was also eliminated. Strikingly, the presence of a unique salt-bridge interaction between E32-R282 in RpoE10 and RpoE10(Mut2) was suggested to directly impact the dynamics of 𝛔2domain. An opposite scenario was observed for NPDKV mutant and truncated SnoaL_2 domain derivative of RpoE10. A schematic diagram of the molecular interaction network between the critical residues of NPDKV, DGGGR, and WLPEP impacting the activation/elimination of promoter activity is presented in Figure 8. We further examined and showed that differential salt-bridge interaction networks could influence the -10 and -35 promoter recognition site through correlated molecular motions. The salt bridge interaction network from N277, D279, K280 and D200, and R204 impacted the conformational alternation in the helix α7 of σ4 domain, which forms the helix-turn-helix motif and interacts with the -35 promoter site, and the “specificity loop” L3 connecting the α2-α3 and forming the -10 recognition cleft. These promoter recognition sites were significantly altered upon mutations in NPDKV and DGGGR. Based on these findings, we suggest that NPDKV and DGGGR motifs are the conformational switches that trigger the productive and unproductive conformations responsible for the activation or elimination of promoter. Also, the significant and differential inward and outward movement of the WLPEP motif makes it a sensor in transmitting the conformational signal from the Snoal_2 domain to2-𝛔4domains. These results suggest that NPDKV and DGGGR, together with WLPEP, may play key roles in modulating correlated motions of the RpoE10 domains. Mutation at the NPDKV motif induces a conformational “switch” of the NPDKV motif to eliminate its inhibitory effect and activate its target promoter.
Overall, our analysis clearly indicates that the NPDKV motif at the C-terminal extension acts as an inhibitory “switch” on RpoE10 activity, and interaction of DGGGR motif with the linker WLPEP motif along with “Arginine tetrad” located between σ2 and σ4, may be required for its activity. In the RpoE10(Del2), however, removal of the C-terminal part of the SnoaL_2 domain, including NPDKV motif as well as DGGGR motif, fails to provide the necessary interaction between WLPEP and DGGGR required for the RpoE10 to acquire the conformation needed for activating abmpromoter (Figure. 2). The above analysis showed that the residues of DGGGR motif interact and stabilize the linker region residues R76 withoutthe 277NPDKV281 motif. The amino acid residues of the277NPDKV281 motif pull the residues of the DGGGR motif away from the linker region and destabilize the interactions of residues of the83WLPEP87 motif. Our simulation results clearly explain the reason for the strong link between NPDKV, DGGGR, and WLPEP motif. Our findings indicated that the conformational transitions associated with the residual motions of NPDKV and DGGGR motif of SnoaL2 domain and WLPEP motif of linker strand connecting the 𝛔2 and 𝛔4 domains could be used to understand the structural alternations and allosteric regulations in ECF41 regulons. Altogether, the results reported in this study will provide a greater understanding of ECF41-associated promoter regulation and will pave a path for understanding the functional role of C-terminal Snoal_2 domain-containing ECF41-𝝈 factors.