Molecular interaction network analyses of NPDKV, DGGGR, and WLPEP motifs
We were keen to understand how, at the molecular level, the DGGGR motif contributes to the enhanced activation of the promoter in both RpoE10(Mut1) and RpoE10(Del1). In contrast, the presence of an intact NPDKV motif has a contrary effect as it eliminates promoter activation. We examined the impact of NPDKV, and DGGGR mutations on the promoter inactivation/activation ability of RpoE10 in the light of molecular interactions, particularly salt-bridge interactions contributed by charged residues N277, D279, and K280 residues of NPDKV motif, and D200 and R204 residues of DGGGR motif across the 200ns MD simulation. These interactions were further mapped onto the representative frame (snapshot) obtained from a most populated cluster from the 200 ns MD simulation data (Figure 6). The cluster was based on the pairwise best-fit RMSDs. The critical point that we focused upon was the molecular interactions that firmly hooked the flexible truncated fragment of Snoal_2 domain of RpoE (Del1) to the linker strand (Figure 5B (inset view) and Figure 6D). Intriguingly, a positively charged Arg74 anchored the negatively charged Asp200 of the DGGG motif in the vicinity of the linker loop. Additionally, an Arg110 strengthens this interaction by holding D200 through the salt bridge (Figure 6D and Supplemental Table S3). The spatial position of R74 is further stabilized by a network of salt-bridges between R74-E77, R74-E109, R74-D194. The aspartic acid dyad, D193-D194, of the remaining part of the SnoaL_2 domain after truncation stabilizes the spatial conformation of R74 via a network of salt-bridges. D193 interacts with R76, R188, H16, while D194 interacts with R74, R76, andR110 through salt-bridges (Supplemental Table S3). Our analysis revealed that this network of salt-bridge interactions pulls and stabilizes Arg73 to form a salt-bridge with highly conserved Asp30 of 30DEAD33motif of helix Ξ±2 of RpoE10-𝛔2 domain. The interaction between R73-D30 was further strengthened by the stacking interaction between R73 and a highly conserved W83, which stays firmly between the Οƒ2-Οƒ4 domains (Figure 6D). In the NPDKV mutant of RpoE10, a scenario similar to RpoE10 (Del1) was observed with a slight variation on the theme. Here, the D30 is anchored on R76 in the vicinity of the W83, as seen in RpoE10(Del1). The R204 of the DGGGR motif forms a salt-bridge interaction with D279 of the NPDKV motif of the wild type RpoE10 (Figure 6A and Supplemental Table S3). However, due to the elimination of N277-R204 interaction in the mutant RpoE10 containing the NAAAV motif, R204 moves towards the linker region and interacts with E77 through salt-bridges (Supplemental Table S3). The DGGGR motif of the wild type RpoE10 contributes to two salt bridges, i.e., D200-K280 and R204-D279 (Figure 6A). However, due to D279A and K280A substitution in RpoE10(Mut1), the D200-K280 and D279-R204 interactions were eliminated (Figure 6C). Consequently, the two positively charged R289 and R292 residues from the SnoaL_2 terminal occupy the spatial location of R204 to form the salt bridge network with D200. Furthermore, D285 strengthens the position of R204 followed by transient salt-bridge interaction of E286 and E290. This interaction network pushes R204 towards the arginine tetrad (R73-R76) near the 𝛔2-linker junction to form the R204-E77 salt bridge. Interestingly, this network of interactions facilitated the R76-D30 salt bridge formation, similar to the case of RpoE10(Del1) (marked by C1, in Figure 6C). Notably, the residues of the Arginine tetrad (R73-R76), W83, and D200 are highly conserved. This suggests that W83 probably serves as an anchor to stabilize the 𝛔2-𝛔4domain of ECF41 bacterial Οƒ factors to activate the promoter. Our analysis indicates that the stable interactions between the DGGGR motif, W83, D30, and (arginine-tetrad) thread the Οƒ2-Οƒ4 domain in a productive orientation and conformation leading to promoter activation.
The next question we pondered was how the NPDKV motif impacts promoter inactivation in the wild type RpoE10 and RpoE10 (Mut2). As evident from Figures 6A and B, the highly conserved D30 and its interaction with R76 of tetrad arginine were eliminated if NPDKV was intact, i.e., RpoE10 and RpoE10 (Mut2). The elimination of the β€œD30-R76” interaction enabled the entire loop of arginine tetrad (R73-R74-R75-R76) to stay away from the W83 containing linker site, thereby attaining a β€œswitch-off” conformation leading to substantial loss of transcription activity (Figure 1). In RpoE10, the K280 and D279 participate in the salt-bridge interaction with D200 and R204, respectively, to restrict the DGGGR motif to attain an unproductive conformation (Figure 6A). Moreover, the side chain of W83 flipped out of the linker towards the NPDKV and DGGGR motif making it no more available for the stacking interaction of D30 and R76.
We were also interested in finding unique interactions in RpoE10 and RpoE10(Mut1), if any, that could shed some light on the observed inactivation and elimination of promoter activity. Indeed, we found a unique salt-bridge, D32-R282, between D32 of 𝛔2-domain and R282 of SnoaL domain that stayed for a longer duration of simulation time (Figure S1 ) and was typical to RpoE10 and RpoE10(Mut2). Out of 160 possible salt-bridges (Supplemental Table S4), we could not find D32-R282 salt-bridge interaction in RpoE10 (Mut1 and Del1). The formation of this unique D32-R282 salt bridge interaction could be attributed to the existence of K280-D200 and K280-E86 salt bridge in RpoE10 (Figure S1 A and B ). Notably, as shown in Figure S1, D32 is located on the outer surface of the helix Ξ±-2, just diagonal to D30, suggesting its impact on the 𝛔2- domain rendering RpoE10 into an unproductive conformation in D32-R282 configuration.