Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal
calcium-binding regulatory protein, has been cleaved by
thrombin to create two fragments, TM1 (1–106) and
TM2 (107–148). NMR and CD results indicate that TM1
and TM2 can associate in the presence of Ca2+
to form a complex similar to native CaM, even though the
cleavage site is not in the linker region between two helix-loop-helix
domains, but rather within an α-helix. Cadmium-113
NMR results show that this complex has enhanced metal-ion
binding properties when compared to either TM1 or TM2 alone.
This complex can bind several CaM-binding target peptides,
as shown by gel bandshift assays, circular dichroism spectra,
and 13C NMR spectra of
biosynthetically methyl-13C-Met-labeled
TM1 and TM2; moreover, gel bandshift assays show that the
addition of a target peptide strengthens the interactions
between TM1 and TM2 and increases the stability of the
complex. Cadmium-113 NMR spectra indicate that the TM1:TM2
complex can also bind the antipsychotic drug trifluoperazine.
However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide
complexes are disrupted by 4 M urea; moreover, TM1 and
TM2 in combination are unable to activate CaM-dependent
enzymes. This suggests that TM1:TM2 mixtures cannot bind
target molecules as tightly as intact CaM, or perhaps that
binding occurs but additional interactions with the target
enzymes that are necessary for proper activation are perturbed
by the proteolytic cleavage. The results presented here
reflect the importance of the existence of helix-loop-helix
Ca2+-binding domains in pairs in proteins such
as CaM, and extend the understanding of the association
of such domains in this class of proteins in general.