Calcium Biochemistry
153
5.1.4
The Penta EF-hand Family
The EF-hand motifs in Ca
2
+
binding
proteins are usually tandemly repeated.
Owing to the recent determination of the
X-ray structure of the small subunits of
the calcium-dependent protease calpain,
it was recognized that also uneven num-
bers of EF-hand domains may exist in
proteins. By comparing the primary struc-
tures of a number of different calcium
binding proteins, Maki et al. identi±ed sev-
eral proteins that, like calpain, contained
±ve EF-hand domains, and therefore intro-
duced the name ‘‘penta EF-hand family’’
or PEF family for this subgroup of EF-
hand-containing proteins. Members of
this family include calpain, sorcin, grancal-
cin, peflin, and ALG-2; the latter was
originally identi±ed as ‘‘
a
poptosis-
l
inked
g
ene’’. Characteristic to all the members
of this family is a two–amino acid in-
sertion into the loop of the ±fth EF-hand
domain, and therefore, at least under phys-
iological conditions, may not bind calcium
anymore. Instead, the ±fth domain may
serve as a dimerization domain, as origi-
nally observed in the calpain structure and
later con±rmed by solving the structure of
ALG-2 and grancalcin.
Proteins of the PEF family have been
quite conserved during evolution; ho-
mologs have been found in invertebrates,
p
lants
,fungi
,andeveninprotists
.Onthe
basis of comparison of the gene structures,
PEF proteins can be classi±ed into two
groups: group I contains ALG-2, peflin,
and their homologs, and group II is com-
posed of calpain, sorcin, and grancalcin.
As indicated before, calpain is a calcium-
dependent, intracellular protease, of which
two major isozymes exist, calpain I and II,
which differ in their Ca
2
+
dependence; that
is, calpain I or
µ
-calpain is activated by mi-
cromolar calcium concentrations, whereas
calpain II or m-calpain needs millimolar
calcium to be activated
in vitro
.B
o
t
h
isoforms exist as heterodimers consisting
of a large subunit (80 kDa), containing
the catalytic site, and of a small regula-
tory subunit (30 kDa). Recently, Suzuki,
Bode et al. determined the structure of
the full-length, heterodimeric human cal-
pain II in its Ca
2
+
-free form as shown
in Fig. 3: the two subdomains IIa and IIb
of the large subunit comprising the cat-
alytic site are rotated against each other,
disrupting the active and the substrate
binding sites, which would explain the
inactivity of calpain in the absence of cal-
cium. Surprisingly, the group of Davies
who determined the structure of calpain
IintheCa
2
+
-bound form suggested that
the cooperative binding of Ca
2
+
to two
non-EF-hand-type Ca
2
+
binding sites de-
rived from two different peptides aligned
the active site cleft and converts the protein
into an active enzyme, and Ca
2
+
binding
to the EF-hand-type binding sites would
contribute to the Ca
2
+
sensitivity of the
enzymes, as documented by mutational
analysis. In addition, both isoforms of cal-
pain are maintained in an inactive form
because of the interaction with an endoge-
nous inhibitor, calpastatin, until external
signals activate the protease.
Increasing evidence indicates that cal-
pain is involved in the regulation of basic
cellular processes such as cell prolifera-
tion, differentiation, and apoptosis owing
to cytoskeletal remodeling, which, under
pathological conditions, could contribute
to tissue damage in heart and brain is-
chemias as well as neurodegeneration in
Alzheimer’s disease.
ALG-2 was originally identi±ed as a gene
linked to apoptosis. It is a 22 kDa highly
conserved and ubiquitously expressed pro-
tein that forms dimers through the ±fth
EF-hand, as documented by the crys-
tal structure. This could provide a new
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