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-π2 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
Ο2-Ο4 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
π2-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 π2-π4domain of ECF41 bacterial Ο factors to activate the promoter. Our
analysis indicates that the stable interactions between the DGGGR motif,
W83, D30, and (arginine-tetrad) thread the
Ο2-Ο4 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 βD30-R76β interaction enabled
the entire loop of arginine tetrad (R73-R74-R75-R76) to stay away from
the W83 containing linker site, thereby attaining a βswitch-offβ
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 π2-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 π2- domain rendering
RpoE10 into an unproductive conformation in D32-R282 configuration.