156
Calcium Biochemistry
family, thereby increasing its Ca
2
+
sensi-
tivity by 3 orders of magnitude. However,
formation of such a complex interferes
with the phosphorylation of annexin II by
different kinases.
5.2.2
Gelsolin
Gelsolin is a multifunctional protein bind-
ing actin in a Ca
2
+
-dependent manner.
It can also nucleate actin polymerization
by binding two actin monomers. Alterna-
tive transcription initiation and selective
RNA processing produces two isoforms
from the same gene; one isoform is
located intracellular and the other extracel-
lular. The intracellular gelsolin is involved
in cell motility–regulating actin function,
whereas extracellular gelsolin can act as
an actin-scavenging system to prevent the
polymerization of actin released after cell
death.
Gelsolin consists of 6 repeat units (G1
to G6) containing 120 to 150 amino acids.
The units are organized in 2 clusters of
similar architecture and are connected
by a flexible linker of about 50 amino
acids that may be cleaved by caspase-3.
One repeat unit, G1, has been crystallized
in a complex with actin. It is organized
in a central four-stranded
β
-sheet motif
faced on one side by a four-turn
α
-helix
parallel to the strands and on the other
side by a shorter
α
-helix perpendicular
to the strands. The longer helix also
serves as the binding partner to a cleft
between two actin subdomains. The G1-
actin-complexed domain comprises two
Ca
2
+
binding sites.
The regulation of gelsolin activity by
Ca
2
+
has recently been proposed. This was
made possible by comparing the Ca
2
+
-
free structure of nonactive gelsolin with
the active, Ca
2
+
-bound gelsolin complexed
to actin. In the absence of Ca
2
+
,t
h
e
six repeat units of gelsolin provide a
very compact globular structure, thus
blocking the actin-binding helices of the
appropriate subdomains. By binding of
Ca
2
+
to gelsolin, the N- and C-terminal
halves of the protein, that is, G1 to G3
and G4 to G6, become separated, thereby
unmasking the actin binding sites, and
hence permit binding to actin strands.
5.2.3
C
2
-domains
Synaptotagmin, a transmembrane pro-
tein of synaptic vesicles, is believed to
act as the major Ca
2
+
sensor of exocy-
tosis and neurotransmitter release. The
cytoplasmic region of synaptotagmin con-
tains two C
2
domains (C
2
Aa
n
dC
2
B)
that are Ca
2
+
binding domains of non-
EF-hand character. These domains have
been ±rst described in protein kinase C by
Nishizuka, and have been suggested to be
responsible for binding to membranes in
response to Ca
2
+
.TheC
2
domain consists
of a
β
-sandwich of two four-stranded
β
-
sheets. The
β
-strands are connected by
loops, some of which bind clusters of
Ca
2
+
ions, primarily through oxygens of
aspartate side chains. Sequence homol-
ogy and structural similarity among C
2
domains of different proteins are high
for the
β
-strands, which probably consti-
tute scaffolds to support the Ca
2
+
binding
loops. A recent detailed mutational analy-
sis of synaptotagmin in
Drosophila
seems
to indicate that only the C
2
Bdomainrepre-
sents the necessary Ca
2
+
sensing domain
required for synaptic vesicle fusion.
6
Systems Controlling Intracellular Ca
2
+
Concentration: Structural and Functional
Properties
Owing to its function as an intracellular
second messenger, the ionized Ca
2
+
has
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