Conformational dynamics of C-terminal partially truncated
RpoE10
The truncated SnoaL_2 domain model of the RpoE10 (Del1) was subjected
to MD simulations for 200 ns trajectories and analyzed to demonstrate
the experimentally observed effect of the DGGGR motif on promoter
activation without NPDKV motif (Figure 1). Strikingly, the truncated
SnoaL_2 domain containing the DGGG motif showed consistent and stable
interactions with the Ο2-Ο4 domain
across the snapshots of the 200ns simulation time (Figure 5 A, B, C;
inset view). Furthermore, we observed stable conformation in the
backbone RMSD of 2, π4 combined
π2-π4 domain, DGGGR, and WLPEP
motifs throughout the simulation (Figure. 5D). In RpoE10 (Del1), after
initial fluctuations in the RMSD, both the π2domain
and combined π2-π4 domain showed an
upsurge in RMSD value at ~125 ns and then stabilized in
remaining ~75 ns (Figure 5D). The RMSD analysis for
π2,4 and
π2-π4domain along with and linker
strand segments suggests that backbone conformations for RpoE10(Del1)
were stable even after partial truncation of the SnoaL_2 domain.
Furthermore, to ensure the stabilized and homogenous conformers, the
histogram of RMSD was plotted against the number of conformers for
RpoE10(Del1). In RpoE10(Mut1), the RMSD for most of the
π2-π4 domain conformers was
restricted to βΌ6-7Γ
(Figure 5E). This analysis suggests that the
truncated SnoaL_2 derivative of RpoE10 containing the DGGG motif adopts
stable conformations of π2-π4domain.
We also evaluated the compactness (Rg value) of the individual
π2,4, combined
π2-π4 domain, and full-length
RpoE10(Del1) model. A steady Rg value for
π2,4 and combined
π2-π4 domain of RpoE10(Del1) was
obtained (Figure 5G) Furthermore, the inter-domain distance motions
between π2 and π4 domains further
mirrors the stable and compact π2-π4domain (Figure 5H). A histogram plot of RMSD against the number of
conformers for RpoE10 (Del1) showed that most of the
π2-π4 domain conformers were
restricted to ~1.76-1.80 nm (Figure. 5F). This analysis
suggests that the truncated SnoaL_2 domain containing the DGGG motif
may constraint the π2-π4 domains to
a stable and compact protein structure required for promoter recognition
and activation. Therefore, the observations that SnoaL_2 domain
constraints Ο domain to a stable and compact structure18 can be attributed to the DGGG motifs of Snoal_2
domain.
We constructed and compared the RMSF plot of RpoE10 (Del1) with RpoE10
and RpoE10 (Mut1) to assess the impact of the DGGGR motif onto the key
residue positions of the truncated SnoaL_2 domain model (Figure 5I). We
focussed on key residue segments essential in initiating the promoter
recognition and determining the ECF Ο factorsβ specificity at the β10
and -35 promoter elements in ECF Ο-dependent promoters31,32. Intriguingly, as shown and marked in Figure 5I,
these key residue positions of the RpoE10 showed contrasting
fluctuations. As compared to RpoE10, an enhanced fluctuation was noticed
in the L3-loop (residues 46-52) of Ο2 in both RpoE10(Del1) and
RpoE10(Mut1). The enhanced flexibility of the L3-loop βspecificity
loopβ is known to favor the -10 promoter recognition32. We also noticed a stable peak in the residues
segment 135-151 of Ο4 domain of RpoE10(Del1) and RpoE10(Mut1), both.
This segment is known to constitute a helix-turn-helix motif that
interacts with the β35 element of the promoter DNA 31(Figure 5I). Therefore, our MD simulation analysis of truncated SnoaL_2
domain, without NPDKV motif, containing the DGGG motif suggests that
promoter recognition and enhanced activation is due to the stable
interactions of DGGG motif with the compact conformations of
π2-π4domain, enhanced flexibility of
the -10 recognizing L3-loop, and stability of the helix-turn-helix motif
that interacts with the β35 element of the promoter DNA.