NMR-STUDIES OF CALDESMON-CALMODULIN INTERACTIONS

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 .