Rg analysis (compactness) of π-domains
The observation that NPDKV and DGGGR motifs of Snoal_2 domain
significantly alter the conformations of2-π4 domain was further evaluated by
measuring the compactness of domains using Radius of gyration (Rg)
analysis of MD simulations. A comparison between the Rg profile and
average Rg value (indicated at the right margin, in Figure 3 A, B, C) of
individual π2, π4, combined
π2-π4 domain, and full-length
RpoE10, RpoE10(Mut1) and RpoE10(Mut2) indicated that the mutation in the
NPDKV motif of SnoaL_2 domain constrains the π2,
π4 and π2-π4domains to a more compact domain structure. A reduced average Rg value
of 1.71 nm for π2-π4 domain of
RpoE10(Mut1) as compared to both RpoE10 (1.75 nm) and RpoE10(Mut2)
(1.73) was observed. In RpoE10(Mut1), although, a sharp fall in the Rg
value from ~1.5 to ~1.25 after 50 ns of
simulation time indicated a rise in the compactness of the
π2 domain (Figure 3B), yet a steady Rg value for
π2-π4 and SnoaL_2 domain suggest a
stable and compact protein. We also determined the minimum distance
between the π2 and π4 domains as a
function of simulation time using theΒ g_mindist Β utility of
GROMACS (Figure 3 D, E, F). A varying degree (0.15-0.22 nm) of
inter-domain distance motions between π2 and
π4 domains in RpoE10 (Mut1) as compared to RpoE10
(0.15-0.17 nm) and RpoE10(Mut2) (0.15-0.17 nm) proteins suggests a
favorable motion for the recognition and activation of promoter.
Furthermore, to zoom in on the differences in the compactness of2-π4 domain, a histogram of RMSD was
plotted against the number of conformers for RpoE10, RpoE10(Mut1), and
RpoE10(Mut2). In RpoE10 (Mut1), the Rg value for
π2-π4 domain conformers was mainly
restricted to ~1.69 nm. However, an increase in the Rg
(~1.73 nm) was noticed for
π2-π4 conformers of both RpoE10 and
RpoE10(Mut2) (Figure 3 G, H and I). The reduced Rg value across a large
number of Ο2-Ο4 conformers in
RpoE10(Mut1) suggests that the mutations in the NPDKV motif of SnoaL_2
domain constrain RpoE10 to a compact and stable structure favoring an
enhanced activation of its promoter.