The binding of the calcium-regulatory protein calmodulin (CaM) to cald
esmon (CaD) contributes to the regulation of smooth muscle contraction
. Two regions of caldesmon have been identified as putative calmodulin
-binding domains. We have earlier reported on the binding of one of th
ese domains to calmodulin (Zhang & Vogel (1994) Biochemistry 33, 1163-
1171). Here we have studied the binding of CaM to synthetic peptides o
f CaD which contain: (1) both the first and second CaM-binding domains
; (2) the second CaM-binding domain; and (3) the sequence between the
first and second CaM-binding domains. Two-dimensional transferred nucl
ear Overhauser enhancement proton NMR measurements as well as circular
dichroism studies of a 22-residue peptide NKETAGLKVGVSSRINEWLTK, whic
h contains the second CaM-binding domain, show that only the C-termina
l half of the peptide becomes cc-helical upon binding to CaM. Somewhat
surprisingly, the shorter 9-residue peptide SRINEWLTK was sufficient
to form a 1:1 complex with CaM; this peptide appears to bind as a 3(10
)-helix. Proton-carbon-13 correlation NMR titration studies with speci
fically labeled [methyl-C-13]methionine CaM were used to study the par
ticipation of the hydrophobic regions in both domains of the dumbbell
shaped CaM in peptide binding. Binding of a 54-residue CaD peptide con
taining both CaM-binding domains affects all the 8 Met residues in the
two hydrophobic domains of CaM (only Met 76 in the linker region of C
aM is not involved), while binding of the second CaM-binding domain of
CaD influences principally Met 51, 71, and Met 124, 144. Simultaneous
binding to CaM of two peptides comprising the first and the second Ca
M-binding domains also caused changes to all Met residues except Met 7
6. Taken together, these data demonstrate that both CaM-binding domain
s of CaD can bind simultaneously to the two hydrophobic regions of CaM
.