Mutations in the NPDKV and DGGGR alter the structural features of the promoter recognition sites
Structural superimposition of ๐›”2 and ๐›”4 domains in RpoE10 with other ๐›”/anti- ๐›” complexes31,32 revealed that in RpoE10, orientation and accessibility of DNA binding surfaces are exposed (Figure 4A). Like its template Mtb-SigJ 18, RpoE10 lacks the first helix ฮฑ1, usually present in the ฯƒ2 domain, which has three helices ฮฑ2โ€“ฮฑ3- ฮฑ4 connected by two loops L2 and L3. Intriguingly, a comparative analysis of the average fluctuations of all the backbone atoms of the amino acid residues (RMSF profile) of the RpoE10, RpoE10 (Mut1), and RpoE10 (Mut2) showed a contrasting pattern at key positions (Figure 4A). The ฮฑ1-helix showed an increased RMSF fluctuation in RpoE10 compared to both RpoE10 (Mut1 and Mut2) (marked as 1 in Figures 4A and B). Remarkably, we noticed a prominent fluctuation in the L3 loop (residues 46-52) between second and third helices (ฮฑ2 andฮฑ3) of RpoE10 (Mut1) (marked as 2 in Figure 4A and B). It has been shown that the flexible โ€œspecificity loopโ€ initiates promoter recognition and determines ECF ฯƒ factorsโ€™ specificity at the โˆ’10 promoter element in ECF ฯƒ-dependent promoters 32. Therefore, a reduced fluctuation in the ฮฑ1 helix and a simultaneously increased fluctuation in the L3 loop of the ๐›”2 domain could favor recognizing -10 promoter and DNA melting by RpoE10 (Mut1), leading to enhanced promoter activation.
Also, in RpoE10, increased fluctuation at the linker-loop junction connecting the ๐›”2 and ๐›”4 domains (peaks are marked as 3 and 4 in Figure 4B), suggests the possibility of conformational instability of ๐›”2 and ๐›”4 domains. As compared to RpoE10(Mut2), the junction region (marked as 4 in Figure 4B) towards the ๐›”4domain is stabilized in RpoE10 (Mut1). Therefore, similar to Mtb-SigJ (18), the decreased fluctuations at the junction of ๐›”2-linker-๐›”4 region depicts the stabilized and tethered ๐›”2 and ๐›”4domains essential for acquiring a productive conformation of RpoE10(Mut1) for enhanced activation of its promoter. Next, we focused on another important segment (135-151)of the ฯƒ4domain, a helix-turn-helix motif known to interact with the โˆ’35 element of the promoter 34. Both, RpoE10 and RpoE10(Mut2) showed an enhanced average fluctuation of backbone atoms of residues 135-151 segment as compared to RpoE10(Mut1) (peaks are marked as 5 in Figure 4 A and B). Unlike RpoE10 and RpoE10(Mut2), the stabilized helix-turn-helix motif of ฯƒ4 domain of RpoE10(Mut1) reasonably favors interaction with the โˆ’35 element of the promoter and, therefore, justifies its enhanced activation.
Interestingly, we notice that the RMSF profile of the RpoE10 (Mut1 and Mut2) showed increased conformational flexibility at both NPDKVand DGGGR motifs (marked as 6 and 9 respectively, in Figure 4B), as compared to that in RpoE10. Since the DGGGR motif is essential for promoter activation (Figure 1), the stabilized conformation of backbone atoms of the NPDKV motif in RpoE10 raises the possibility to obstruct the DGGGR motif away from the ๐›”2-๐›”4 linker site. In this situation, the stabilized orientation of the NPDKV motif may lead to the formation of unproductive conformations of2-๐›”4 domain in RpoE10, and therefore a possible reason for the elimination of its activity. Notably, in RpoE10, another segment of 230-238 residues (marked as 8 in Figures 4A and B) showed a sharp and distinct rise in the fluctuations of backbone atoms compared to RpoE10(Mut1 and Mut2). This segment forms the core of the SnoaL_2 domain, and therefore the backbone fluctuations may act as a trigger signal for eliminating the promoter activation of RpoE10.
RMSF analysis showed that the NPDKV mutant form of RpoE10 containing an intact DGGGR motif showed stable conformations at the -35 promoter binding site (residues 135-151), enhanced flexibility of L3 โ€œspecificity loopโ€ (residues 45-52) around the -10 promoter recognition site, and stabilized linker region connecting the ๐›”2-๐›”4 domain which are essential features to attain a productive conformation for enhanced promoter activation.