CALMODULIN-BINDING OF A PEPTIDE DERIVED FROM THE REGULATORY DOMAIN OFBORDETELLA-PERTUSSIS ADENYLATE-CYCLASE

This paper reports the solution conformation and calmodulin binding of a 43-residue peptide from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. The peptide (P-225-267) was synthesized and N-15-labeled at specific amino acids. It binds calmodulin with an equilibrium dissociation constant of 25 nM. Assignment of the NMR spec trum of the free peptide and analysis of the NOE connectivities and se condary shifts of C alpha protons allowed us to identify a 10-amino ac id fragment (Arg(237) to Arg(246)) which is in rapid equilibrium betwe en alpha-helical and irregular structures. Titration experiments showe d that at substoichiometric molar ratios the two molecules are in inte rmediate exchange between free and bound conformations. Using N-15-edi ted methods we assigned a large part of resonances of the labeled resi dues in the bound peptide. Analysis of the chemical shift differences between free and bound states shows that the fragment Leu(240)-Ala(257 ) is the most affected by the interaction. The proton spectra of the c almodulin, in the free and complexed states were extensively assigned using homonuclear experiments. Medium- and long-range NOE patterns are consistent with a largely conserved secondary and tertiary structure. The main changes in chemical shift of calmodulin resonances are group ed in six structural regions both in NH2- and COOH-terminal domains. I ntermolecular NOE connectivities indicate that the NH2-terminal of the bound peptide fragment is engulfed in the COOH-terminal domain of cal modulin. The interaction geometry appears to be similar to those previ ously described for myosin light chain kinase or calmodulin kinase II fragments.