A Direct Test of the Reductionist Approach to Structural Studies of Calmodulin Activity: RELEVANCE OF PEPTIDE MODELS OF TARGET PROTEINS

James K Kranz,Eun K Lee,Angus C Nairn,A Joshua Wand

doi:10.1074/jbc.c200139200

James K Kranz, Eun K Lee + Show 2 more

Open Access

https://doi.org/10.1074/jbc.c200139200

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Abstract

Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM. CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM. CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM. CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.

Highlights

From the ‡The Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059 and the ¶Laboratory of Molecular and Cellular Neurosciences, The Rockefeller University, New York, New York 10021
Using NMR techniques optimized for large proteins, backbone resonance assignments were determined for CaM bound to the intact CaM-dependent protein kinase I (CaMKI) enzyme
Chemical shifts of CaM bound to a peptide corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are compared with the CaM1⁄7CaMKI complexes

Summary

Introduction

Ca2؉-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR 13C15N-1H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM1⁄7CaMKIp complex indicate large differences throughout the CaM sequence.

Results

Conclusion