Neurogranin alters the structure and calcium binding properties of calmodulin.
Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca(2+) but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca(2+) binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM.
From the Departments of Neurobiology and Anatomy and.
Laurel
L
Hoffman
From the Departments of Neurobiology and Anatomy and.
Anuja
A
Chandrasekar
Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030.
Xu
X
Wang
Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030.
John A
JA
Putkey
From the Departments of Neurobiology and Anatomy and m.n.waxham@uth.tmc.edu.
M Neal
MN
Waxham
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D051579:Neurogranin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D002147:Calmodulin; D051579:Neurogranin; D002118:Calcium
Calcium; Calmodulin; Cell Signaling; Intrinsically Disordered Proteins; Isothermal Titration Calorimetry; Kinetics; Nuclear Magnetic Resonance; Protein Structure; Protein-Protein Interactions; Signal Transduction