A mechanism for calmodulin (CaM) trapping by CaM-kinase II defined by a family of CaM-binding peptides.
Autophosphorylation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) induces a striking >1,000-fold increase in its affinity for CaM, which has been called CaM trapping. Two peptides modeled after the CaM binding domain of CaM-kinase II were previously shown to kinetically resemble CaM binding to phosphorylated and dephosphorylated forms of the enzyme, thus providing a model system with which to define the molecular basis of CaM trapping. In this report, the specific contribution of each amino acid to the rates of association and dissociation, and the overall Kd of CaM binding to CaM-kinase II was determined using an overlapping peptide family, and a fluorescently labeled CaM. The association rate constants were similar for the entire family of peptides and ranged from 8 x 10(7) to 32 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for the peptides varied by >3500-fold and ranged from 0.26 to 7 x 10(-5) s-1. These rate constants yield overall Kd values for binding CaM to the peptides that range from 2 x 10(-9) M to 2 x 10(-13) M. Extending the low affinity CaM-binding peptide, CKII(296-312), to include 293Phe-Asn-Ala295 provided the single largest contribution to the decreased dissociation rate constant, 1,300-fold. It was further shown using Ala-substituted peptides that the basic residues 296Arg-Arg-Lys299 were also essential for slow CaM dissociation; however, their contribution was realized only when 293Phe-Asn-Ala295 were present. These results suggest a plausible model in which autophosphorylation of CaM-kinase II leads to a conformational change in the region of 293Phe-Asn-Ala295 which makes these residues accessible for binding to CaM. As a consequence of these changes, further CaM contacts with 296Arg-Arg-Lys299 are established leading to high affinity CaM binding or "CaM trapping."
Waxham
M N
MN
Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, Houston, Texas 77030, USA.
Tsai
A L
AL
Putkey
J A
JA
D019943:Amino Acid Substitution; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013816:Thermodynamics
D002147:Calmodulin; D009842:Oligopeptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases