144
Calcium Biochemistry
5
Intracellular Calcium Binding Proteins
5.1
The EF-hand Protein Family
As pointed out before, the concentration
of the intracellular Ca
2
+
in eucaryotic
cells is closely regulated to remain below
5
×
10
7
M in a resting cell, whereas
outside cells [Ca
2
+
]is10
3
M, resulting in
a steep concentration gradient across the
plasma membrane. The intracellular level
of Ca
2
+
can transiently increase owing
to a response to extracellular signals. In
order to function as a second messenger,
the Ca
2
+
signal is transduced by a variety
of Ca
2
+
binding proteins. In contrast
to
the
extracellular,
low-afFnity
Ca
2
+
binding proteins, the intracellular proteins
bind Ca
2
+
with high afFnity owing to
a sequential arrangement of the amino
acids ligating calcium in a loop flanked
by two helical segments. This common
helix-loop-helix Ca
2
+
binding motif, also
known as the E± hand as coined by
Kretsinger, is an important entity of the
intracellular Ca
2
+
receptor proteins for
triggering cellular responses. The helix-
loop-helix motif can be present in several
copies in these proteins. The number of
the latter is steadily increasing, (to date
more than 600 have been described, but
for most of them a precise function is not
known yet).
In 1973, Kretsinger and Nockolds re-
ported the crystal structure of parvalbu-
min. The protein consists of two homol-
ogous calcium binding domains each of
which contains two
α
-helices perpendicu-
lar to each other enclosing a Ca
2
+
binding
loop, the well-known ‘‘E±-hand’’. Parvalbu-
min thus became the prototype of numer-
ous Ca
2
+
binding proteins with similar
structural properties, such as calmodulin,
troponin C, recoverin, S100 proteins, and
others. To date, more than 50 struc-
tures of E±-hand proteins are known.
All E±-hand domains show a pentagonal-
bipyramidal coordination of the Ca
2
+
ion
in the loop flanked by the two approxi-
mately perpendicular helices. The residues
forming the ligands to Ca
2
+
are highly con-
served within a contiguous sequence of 12
residues spanning the loop and the begin-
ning of the second helix. Loop residues in
positions1
,3
,5
,and12contributemono-
dentate (1, 3, 5) or bidentate (12) Ca
2
+
ligands through side-chain oxygens and
residue 7 through its backbone carbonyl
oxygen. Therefore, an invariant glycine
residue is in position 6 to permit the sharp
bend necessary to ligate Ca
2
+
through the
oxygen of a side chain (5) and a backbone
carbonyl (7). Residue 9 provides an addi-
tional ligand, either directly through an
oxygen of its side chain or indirectly via a
water molecule. In position 1 of the loop,
usually an aspartate is located, whereas in
position 12, glutamic acid is invariant. The
latter has two reasons:
1. both oxygens of the side-chain carboxy-
late ligate to calcium and
2. glutamic
acid
has
α
-helix-inducing
propensity.
The Ca
2
+
binding domains usually
occur in pairs stabilized by hydrogen-
bond bridges between the central residues
of adjacent loops that form a minimal
antiparallel
β
-sheet. Owing to these pair-
forming Ca
2
+
binding domains, the Ca
2
+
afFnity to these sites and the cooperativity
of binding is enhanced. Calmodulin and
troponin C contain four Ca
2
+
binding
sites. Both proteins display Ca
2
+
binding
characteristics compatible with a ‘‘pair-of-
pairs’’ model of E±-hands, that is, the two
globular domains at the N- and C-terminal
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