EF
LF
The active
site residues in calpain are mis-aligned
in the apo, Ca2+-free form. Alignment for catalysis
requires binding of Ca2+ to two non-EF-hand sites, one in each of the core
domains I and II. Using domain swap constructs between the protease cores of
the - and m- isoforms, (which have different Ca2+
requirements), and structural and biochemical characterization of site-directed
mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to
the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding
to this domain as shown by the crystal structure of D106A I-II. However, at
elevated Ca2+ concentrations this mutant still forms the double salt bridge
that links the two Ca2+ sites and becomes nearly as active as I-II. Elimination
of the bridge in E333A I-II has a more drastic effect on enzyme action, especially
at low Ca2+ concentrations. Domain II Ca2+ binding appears
essential because Ca2+ coordinating side-chain mutants E302R and D333A have
severely impaired I-II activation and activity. The introduction of mutations
into the whole heterodimeric enzyme that eliminate
the salt bridge or Ca2+ binding to domain II produce similar phenotypes,
suggesting that the protease core Ca2+ switch is crucial and cannot be
overridden by Ca2+ binding to other domains.
