To
model the possible dynamic fluctuations that might be associated with
this putative allosteric pathway, we carried out Anisotropic Network
Modeling (ANM) (40, 41). ANM is based on a Gaussian network that
considers protein structures as elastic networks in which the nodes
correspond to the Cα atoms, connected by identical spring constants and
in which a Kirchhoff matrix is used to represent the topology of
internal contacts. ANM has been used to successfully model the known
dynamical modes of the allosteric transition in hemoglobin (49). The
three most prominent normal modes obtained from the ANM calculation onA. fulgidus DsrAB reveal significant motion of one heterodimer
with respect to the other, in addition to internal modes within monomers
and heterodimers (Figure 7, Movies S1-S4). The red squares along the
diagonal in the correlation matrix (Figure S5) indicate motions within
each domain of each subunit. Within subunits, individual domains exhibit
anti-correlated motions (blue). Off diagonal correlations (red) are
observed between subunits within a heterodimer and between heterodimers.
For example, motions of domain 1 of the DsrA1 subunit are correlated
with the ferredoxin domain of the DsrB1 subunit (Figure S5, yellow
circles) and with the domain 1 of the DsrB2 subunit (Figure S5, green
circle). The largest motions as evaluated by the calculated B-factors
(Figure S6), were observed in the slowest mode for the ferredoxin
domains of the DsrA1/2 and DsrB1/2 subunits, and in a region of the
DsrB1/2 subunits that is near the structural heme and adjacent to one of
the FPEC heme road residues, N180B. The point of contact between the two
heterodimers is the FPEC heme road residue, T351. It corresponds to a
pivot point (low B-factor) for the most significant modes.
Interestingly, the shifts in the backbone observed between the two
models of the MV2-Eury sequence, particularly apparent in the ferredoxin
domains (Figure 6), mimic the rocking like conformational changes
associated with the first two normal modes (Figure 7).
In parallel, we resumed the crystallographic refinement using current
Phenix software to evaluate the impact of simulating more numerous but
smaller segments for TLS refinement of the Archeoglobus fulgidusDsrAB crystal structure, PDB3MM5 (20). Changing from two to twenty-one
TLS groups did not affect significantly Rwork/Rfree factors (shifting
from 15.8 to 16.1 and from 18.8 to 18.5, respectively) and did not yield
a much nicer electron density for one highly flexible heterodimer while
the other one is very well resolved and mainly rigid. This result
suggested that the overall flexibility relates mainly to rigid body
movement of the whole heterodimers. Our ensemble refinement based on 34
conformational states slightly improves the agreement with diffraction
data (Rwork: 15.3 from 16.1 and Rfree: 18.5 from 18.5) and suggested
some flexibility does occur in the crystal state. Consistent with the
notion of dynamic displacements of one heterodimer relative to the
other, ensemble refinement of the 3mm5 crystal structure yielded one
highly resolved heterodimer (but for only small loops actually
flexible), while the second appeared more flexible as a whole with some
external segments appearing poorly resolved (see Fig S7). This
asymmetric behavior relates to the odd crystal packing that is almost
absent for the flexible heterodimer while crystal organization relies on
contacts involving the other apparently more rigid heterodimer. The
B-factors recapitulate the same picture with one ‘cold’ heterodimer and
the second heterodimer getting ‘hotter’ from their common interface to
its outward surface..