  This study reveals the essence of ligand recognition mechanisms by which
calmodulin (CaM) controls a variety of Ca2+ signaling processes. We study eight
forms of calcium-loaded CaM each with distinct conformational states. Reducing
the structure to two degrees of freedom conveniently describes main features of
conformational changes of CaM via simultaneous twist-bend motions of the two
lobes. We utilize perturbation-response scanning (PRS) technique, coupled with
molecular dynamics simulations to analyze conformational preferences of
calcium-loaded CaM, initially in extended form. PRS is comprised of sequential
application of directed forces on residues followed by recording the resulting
coordinates. We show that manipulation of a single residue, E31 located in one
of the EF hand motifs, reproduces structural changes to compact forms, and the
flexible linker acts as a transducer of binding information to distant parts of
the protein. Independently, using four different pKa calculation strategies, we
find E31 to be the charged residue (out of 52), whose ionization state is most
sensitive to subtle pH variations in the physiological range. It is proposed
that at relatively low pH, CaM structure is less flexible. By gaining charged
states at specific sites at a pH value around 7, local conformational changes
in the protein will lead to shifts in the energy landscape, paving the way to
other conformational states. These findings are in accordance with FRET
measured shifts in conformational distributions towards more compact forms with
decreased pH. They also corroborate mutational studies and proteolysis results
which point to the significant role of E31 in CaM dynamics.
