Experimentally exploring the conformational space sampled by domain reorientation in calmodulin

The conformational space sampled by the two-domain protein
calmodulin has been explored by an approach based on four sets
of NMR observables obtained on Tb3+and Tm3+substituted
proteins. The observables are the pseudocontact shifts and residual
dipolar couplings of the C-terminal domain when lanthanide substitution
is at the N-terminal domain. Each set of observables
provides independent information on the conformations experienced
by the molecule. It is found that not all sterically allowed
conformations are equally populated. Taking the N-terminal domain
as the reference, the C-terminal domain preferentially resides
in a region of space inscribed in a wide elliptical cone. The axis of
the cone is tilted by ≈30° with respect to the direction of the
N-terminal part of the interdomain helix, which is known to have
a flexible central part in solution. The C-terminal domain also
undergoes rotation about the axis defined by the C-terminal part
of the interdomain helix. Neither the extended helix conformation
initially observed in the solid state for free calcium calmodulin nor
the closed conformation(s) adopted by calcium calmodulin either
alone or in its adduct(s) with target peptide(s) is among the most
preferred ones. These findings are unique, both in terms of structural
information obtained on a biomolecule that samples multiple
conformations and in terms of the approach developed to achieve
the results. The same approach is in principle applicable to other
multidomain proteins, as well as to multiple interaction modes
between two macromolecular partners.