title: Characterisation of the interaction of the C-terminus of the dopamine D2 receptor with neuronal calcium sensor-1.
abstract: NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ∼14.3 µM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.
authors: NMR Centre for Structural Biology, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom. Lu-Yun.Lian@liv.ac.uk
authors: Lu-Yun
authors: LY
authors: Lian
authors: Sravan R
authors: SR
authors: Pandalaneni
authors: Pryank
authors: P
authors: Patel
authors: Hannah V
authors: HV
authors: McCue
authors: Lee P
authors: LP
authors: Haynes
authors: Robert D
authors: RD
authors: Burgoyne
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051580:Neuronal Calcium-Sensor Proteins / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D017448:Receptors, Dopamine D2; C101464:frequenin calcium sensor proteins