Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

152 Trewhella and Krueger
P(r) function derived for CaM within the complex shows that it undergoes an
unhindered conformational collapse upon binding MLCK that is indistinguishable
from that observed with the isolated CaM-binding peptides. This result
requires that the CaM-binding domain, as well as some portion of its neighboring
residues in the sequence be completely removed from interactions with the
catalytic core. The model further shows that CaM binds to the enzyme at a site
that is distant from the catalytic cleft, which also requires a significant movement
of the autoinhibitory sequence away from the surface of the catalytic
core. These data provided the first direct structural evidence for the
autoinhibitory hypothesis for MLCK activation.
The neutron experiments further revealed that the binding of a peptide substrate
and a nonhydrolyzable analog of ATP (AMP.PNP) to the 4Ca 2+ /CaMbound
kinase results in a “closure” of the enzyme’s catalytic cleft, thus bringing
together all the elements required for catalysis. In addition, the separation of
the centers-of-mass of the CaM and MLCK components is shortened (by
approx 12 Å bringing CaM closer to the catalytic site. Finally, there is a reorientation
of CaM with respect to the kinase upon substrate binding that results
in interactions between the N-terminal sequence of CaM and the kinase that
were not observed in the complex without substrates. This reorientation is of
particular interest in light of the observation that deletion of the N-terminal
leader sequence of CaM abolished CaM-dependent activation of skeletal
muscle MLCK although having no effect on the apparent affinity (20). The
neutron-derived models thus provide evidence that there is an interaction
between the N-terminal helix of CaM and the surface of the kinase that is
important for activation.
Small-angle X-ray scattering has recently provided evidence for a 2Ca 2+ /
CaM/MLCK intermediate in the MLCK activation mechanism (21). Analysis
of the forward scattering, or I 0, data from a Ca 2+ titration of CaM/MLCK complexes
showed that there is full complex formation when there is only two mole
equivalents of Ca 2+ per complex. This 2Ca 2+ intermediate had been suggested
to exist under physiological conditions in the absence of a Ca 2+ signal (22,23).
The purpose of such an intermediate could be to restrain the CaM from diffusing
away in the absence of the Ca 2+ signal in rapidly cycling functions such as
muscle contraction and relaxation. Because the Ca 2+ affinities of CaM are
strongly affected by its different target binding sequences, it has been further
suggested that CaM-binding sequences in different enzymes may serve the
purpose of “tuning” the calcium affinities of the Ca 2+ -binding sites so as to
optimize for the formation of such intermediates when needed. Thus, in the
CaM/MLCK example, we see the C-terminal domain of CaM may in fact
be functioning as an “anchor” whereas CaM’s N-terminal lobe would possess
the regulatory function, alternately binding and releasing the autoinhibitory
sequence of MLCK in response to the Ca 2+ signal.