Circular Dichroism and 1H NMR Studies on the Structures of Peptides Derived from the Calmodulin-binding Domains of Inducible and Endothelial Nitric-oxide Synthase in Solution and in Complex with Calmodulin

There exist two types of nitric-oxide synthase (NOS); constitutive isozymes that are activated by binding calmodulin in response to elevated Ca2+ and an inducible isozyme that binds calmodulin regardless of Ca2+. To study the structural basis of the difference in Ca2+ sensitivity, we have designed synthetic peptides of minimal lengths derived from the calmodulin-binding domain of endothelial NOS (eNOS) and that of macrophage NOS (iNOS). A peptide, KRREIPLKVLVKAVLFACMLMRK, derived from human iNOS sequence, retained the ability to bind to calmodulin both in the presence and absence of Ca2+, while a peptide derived from human eNOS sequence, RKKTFKEVANAVKISASLMG, bound to calmodulin only in the presence of Ca2+. Circular dichroism and two-dimensional 1H nuclear magnetic resonance studies suggested that both peptides assume nascent α-helical structures in aqueous solution. When mixed with calmodulin, both peptides showed circular dichroism spectra characteristic for α-helix. In contrast to other target proteins, the addition of iNOS peptide to calmodulin did not affect the Ca2+ binding of calmodulin appreciably. The peptide derived from the calmodulin-binding domain of iNOS, therefore, binds in α-helical structures both to Ca2+-calmodulin and apo-calmodulin, which is unique among various target proteins of calmodulin.