Calcium Biochemistry
147
half forms an
α
-helix, the C-terminal half
is a hairpin loop folding back on itself.
In addition, the peptide is oriented in a
parallel fashion relative to the two halves
of CaM.
Interestingly, recent experiments with
small-angle X-ray scattering and neutron
scattering by the group of Trewhella pro-
vided evidence that also when CaM is
complexed to an intact enzyme, that is,
MLCK, CaM undergoes a conformational
collapse identical to that observed with
the peptide corresponding to its bind-
ing domain.
The Frst crystal structure of a CaM-
dependent enzyme, CaM-dependent ki-
nase I (CaMKI), obtained in the absence
of CaM, that is, in the autoinhibited state,
supports the view that the C-terminal reg-
ulatory domain forms a helix-loop-helix
segmen
ttha
tin
te
r
fe
resw
i
ththetwodo
-
mains of the catalytic core, that is, with
the binding site of peptide substrates and
with the ATPbinding site. An interesting
feature of this structure is the striking
observation that the N-terminal part of
the CaM binding domain is accessible
for an initial interaction with CaM. This
part comprises the loop region of the reg-
ulatory domain including the conserved
tryptophan that provides the hydropho-
bic anchor for the C-terminal half of
CaM. ±urthermore, it was recently pos-
tulated that the interaction between the
C-terminal half of CaM and the CaM bind-
ing domain of a CaM-dependent enzyme
is necessary, and sometimes already sufF-
cient, to release the autoinhibited state of
the enzyme. This view was based on the
three-dimensional NMR structure of the
complex between CaM and the N-terminal
part of the binding domain of the plasma
membrane calcium pump (±ig. 2b), a view
that is supported by recent structural stud-
ies on MLCK indicating that initial binding
of CaM to MLCK occurs already at substoi-
chiometric Ca
2
+
concentrations, that is, at
about 2 moles of Ca
2
+
/mol of CaM.
Calmodulin is a very versatile modula-
tor protein interacting with a variety of
different enzymes such as adenylate cy-
clases, cyclic nucleotide phosphatases, NO
synthases, protein kinases, and others.
One of the best-studied families of CaM-
dependent enzymes is the CaM-dependent
protein kinases, which will be outlined in
more detail.
5.1.1.2
Calmodulin-dependent Kinases
Calmodulin-dependent
protein
kinases
can be divided into two categories
1. Kinases dedicated to single substrates,
called
monosubstrate kinases
.Examp
les
of this class are the myosin light chain
kinase (MLCK), the phosphorylase ki-
nase, and CaM-dependent kinase III or
elongation factor 2 kinase. MLCK will
be described in more detail.
2. Kinases dedicated to different sub-
strates, called
multisubstrate kinases
.Ex-
amples of this class are CaM kinase I,
II, and IV, and the CaM kinase kinase,
activating CaMKI and IV.
5.1.1.2.1
Monosubstrate CaM Kinases
Myosin Light Chain Kinase (MLCK)
The
most
extensively
studied
calmodulin-
dependent enzyme is the myosin light
chain kinase (MLCK). This enzyme cat-
alyzes the phosphorylation of a speciFc
serine residue in the N-terminal portion of
the myosin II regulatory light chains. Two
types of MLCK exist that differ genetically,
biochemically, and physiologically and that
are expressed in two different types of
muscles: the MLCK of skeletal muscles
(skMLCK) and of smooth or nonmuscle
cells (smMLCK). The two different MLCK
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