Androcam is a testis-specific protein of Drosophila
melanogaster, with 67% sequence identity to calmodulin
and four potential EF-hand calcium-binding sites. Spectroscopic
monitoring of the thermal unfolding of recombinant calcium-free
androcam shows a biphasic process characteristic of a two-domain
protein, with the apo-N-domain less stable than the apo-C-domain.
The two EF hands of the C-domain of androcam bind calcium
cooperatively with 40-fold higher average affinity than
the corresponding calmodulin sites. Magnesium competes
with calcium binding [Ka(Mg) ∼3
× 103 M−1]. Weak
calcium binding is also detected at one or more N-domain
sites. Compared to apo-calmodulin, apo-androcam has a smaller
conformational response to calcium and a lower α-helical
content over a range of experimental conditions. Unlike
calmodulin, a tryptic cleavage site in the N-domain of
apo-androcam remains trypsin sensitive in the presence
of calcium, suggesting an altered calcium-dependent conformational
change in this domain. The affinity of model target peptides
for androcam is 103–105 times
lower than for calmodulin, and interaction of the N-domain
of androcam with these peptides is significantly reduced.
Thus, androcam shows calcium-induced conformational responses
typical of a calcium sensor, but its properties indicate
calcium sensitivity and target interactions significantly
different from those of calmodulin. From the sequence differences
and the altered calcium-binding properties it is likely
that androcam differs from calmodulin in the conformation
of residues in the second calcium-binding loop. Molecular
modeling supports the deduction that there are significant
conformational differences in the N-domain of androcam
compared to calmodulin, and that these could affect the
surface, conferring a different specificity on androcam
in target interactions related to testis-specific calcium
signaling functions.