Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium.
The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin. Analysis of observed chemical shifts and patterns of NOEs on the main chain indicates extensive and regular secondary structure throughout the N-terminal domain. In contrast, the helices of the C-terminal domain are somewhat irregular and are dynamically averaged. The EF-hands are intact in the N-terminal domain with the loops forming a short antiparallel beta sheet. Under low-salt conditions, two helix-loop-helix EF-hand motifs are present in the C-terminal domain of apoCaM but do not show interstrand NOEs. The spectral perturbations of apoCaM upon complexation with the Neurop peptide are relatively small with the larger chemical shift perturbations occurring in the C-terminal domain. The general secondary structure and tertiary organization appears to remain roughly the same as in free apoCaM. Stoichiometric titration of the apoCaM.Neurop complex with calcium indicates that the C-terminal domain EF-hands have a higher affinity for calcium than N-terminal domain EF-hands. Thus, this complex offers a unique opportunity to examine the structural and energetic consequences of calcium-dependent and calcium-independent binding of peptide to calmodulin.
Urbauer
J L
JL
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Short
J H
JH
Dow
L K
LK
Wand
A J
AJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D019922:GAP-43 Protein; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D017433:Protein Structure, Secondary
D002147:Calmodulin; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D011189:Potassium Chloride; D002118:Calcium