The physiological role of the skeletal muscle-specific
calpain 3, p94, is presently unknown, but defects in its gene cause
limb girdle muscular dystrophy type 2A. This
calcium-dependent
cysteine protease resembles the large subunit of
m-calpain but with three unique additional sequences: an N-terminal region (NS), and two insertions (IS1 and IS2). The latter two insertions have been linked to the chronic instability of the whole
enzyme both in vivo and in vitro. We have shown previously that the core of p94 comprising NS, domains I and II, and IS1 is stable as a
recombinant protein in the absence of Ca(2+) and undergoes
autolysis in its presence. Here we show that p94I-II cannot hydrolyze an exogenous substrate before
autolysis but is increasingly able to do so when
autolysis proceeds for several hours. This gain in activity is caused by cleavage of IS1 during
autolysis because a deletion mutant lacking the NS region (p94I-II DeltaNS) shows the same activation profile. Similarly, the
calpain inhibitors E-64 and
leupeptin have almost no inhibitory effect on substrate hydrolysis by p94I-II soon after
calcium addition but cause complete inhibition when
autolysis progresses for several hours. As
autolysis proceeds, there is release of the internal IS1
peptide, but the two portions of the core remain tightly associated. Modeling of p94I-II suggests that IS1 contains an amphipathic alpha-helix flanked by extended loops. The latter are the targets of
autolysis and limited digestion by exogenous
proteases. The presence and location of the alpha-helix in recombinant IS1 were confirmed by circular dichroism and by the introduction of a L286P helix-disrupting mutation. Within p94I-II, L286P caused premature autoproteolysis of the
enzyme. IS1 is an elaboration of a loop in domain II near the active site, and it acts as an internal autoinhibitory propeptide, blocking the active site of p94 from substrates and inhibitors.