title: Solution NMR structure of the Ca2+-bound N-terminal domain of CaBP7: a regulator of golgi trafficking.
abstract: Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca(2+) binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca(2+) but not Mg(2+) and undergoes significant conformational changes in both secondary and tertiary structure upon Ca(2+) binding. The Ca(2+)-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca(2+)-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.
authors.affiliation: Physiological Laboratory, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, United Kingdom.
authors.forename: Hannah V
authors.initials: HV
authors.lastname: McCue
authors.forename: Pryank
authors.initials: P
authors.lastname: Patel
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Herbert
authors.forename: Lu-Yun
authors.initials: LY
authors.lastname: Lian
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
authors.forename: Lee P
authors.initials: LP
authors.lastname: Haynes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015778:Minor Histocompatibility Antigens; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013499:Surface Properties
chemical_list: C540140:CABP7 protein, human; D002135:Calcium-Binding Proteins; D015778:Minor Histocompatibility Antigens; D012996:Solutions; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D002118:Calcium