Abstract
AKAP79/150 is essential for coordinating second messenger-responsive enzymes in processes including synaptic long-term depression. Ca2+ directly regulates AKAP79 through its effector calmodulin (CaM), but the molecular basis of this regulation was previously unknown. Here, we report that CaM recognizes a ‘1-4-7-8’ pattern of hydrophobic amino acids starting at Trp79 in AKAP79. Cross-linking coupled to mass spectrometry assisted mapping of the interaction site. Removal of the CaM-binding sequence in AKAP79 prevents formation of a Ca2+-sensitive interface between AKAP79 and calcineurin, and increases resting cellular PKA phosphorylation. We determined a crystal structure of CaM bound to a peptide encompassing its binding site in AKAP79. CaM adopts a highly compact conformation in which its open Ca2+-activated C-lobe and closed N-lobe cooperate to recognize a mixed α/310 helix in AKAP79. The structure guided a bioinformatic screen to identify potential sites in other proteins that may employ similar motifs for interaction with CaM.
Highlights
AKAP79/150 is essential for coordinating second messenger-responsive enzymes in processes including synaptic long-term depression
Constitutive binding sites for calcineurin and protein kinase (PKA) have been mapped within AKAP79: PKA binds to an amphipathic helix near to the anchoring protein’s C-terminus15, whereas calcineurin binds to the sequence PIAIIITD between AKAP79 residues 337 and 34319
To determine which lysines had cross-linked within the complex, the sample was digested with trypsin, and cross-linked peptides were identified using liquid chromatography (LC)-MS/MS
Summary
AKAP79/150 is essential for coordinating second messenger-responsive enzymes in processes including synaptic long-term depression. Calmodulin (CaM) is a Ca2+-sensing protein that is expressed in all eukaryotic cells It mediates many essential processes driven by Ca2+, including long-term changes in synaptic connections in the brain, apoptosis, and immune responses. High-resolution crystal and NMR structures have established how both lobes of CaM act in concert to coordinate either 1-5-10 or 1-8-14 class motifs. High-resolution crystal and NMR structures have established how both lobes of CaM act in concert to coordinate either 1-5-10 or 1-8-14 class motifs7,8 Outside of these predominant classes, in a few cases Ca2+/CaM binds in an extended conformation to longer recognition sequences. Despite decades of progress in understanding CaM recognition sequences, CaM-binding sites in some Ca2+/CaM-regulated proteins have eluded mapping One such protein is AKAP79, which was the focus of this investigation. The interlinking pattern between CaM and AKAP79 served as a foundation for identifying and characterizing the CaM-binding site in AKAP79
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