Abstract
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 alpha-helical structures in aqueous solution. When mixed with calmodulin, both peptides showed circular dichroism spectra characteristic for alpha-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 alpha-helical structures both to Ca2+-calmodulin and apo-calmodulin, which is unique among various target proteins of calmodulin.
Highlights
A large number of the binding domains of calmodulin-binding proteins have been shown to have a so-called basic amphiphilic ␣-helix as a common motif [13, 14]
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 endothelial NOS (eNOS) sequence, RKKTFKEVANAVKISASLMG, bound to calmodulin only in the presence of Ca2؉
Design of Peptides Derived from eNOS and iNOS Sequences Suitable for Structural Studies—The amino acid sequences of the peptides synthesized in the present study are shown together with those reported in the previous studies (Table I)
Summary
This study 22 papers have recently appeared demonstrating the interaction of peptides derived from the putative iNOS calmodulin-binding domain with calmodulin [22, 25,26,27], all of the synthetic peptides suffer from the formation of large aggregates of calmodulin and peptides, which makes them unsuitable for detailed structural studies. We have designed synthetic peptides of minimal length derived from the calmodulin-binding domain of human iNOS and eNOS, which still retained the characteristics of the intact proteins in calmodulin binding but would not cause aggregation of calmodulin-peptide complexes. This enabled us to study their structures in solution and in calmodulin complex in detail. The peptide derived from the iNOS sequence formed calmodulin complexes in ␣-helical conformation both with apo- and Ca2ϩ-calmodulin
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