title: The C2A-C2B linker defines the high affinity Ca(2+) binding mode of rabphilin-3A.
abstract: The Ca(2+) binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca(2+) binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca(2+)-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca(2+) ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca(2+) binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca(2+)-bound state of the C2B domain. In addition, this Ca(2+) binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca(2+) binding mode not only shows how a C2 domain increases its intrinsic Ca(2+) affinity, but also provides the structural base for an atypical protein-Ca(2+)-phospholipid binding mode of rabphilin-3A.
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, and the University of Göttingen, 37077 Göttingen, Germany.
authors.forename: Pierre
authors.initials: P
authors.lastname: Montaville
authors.affiliation: Department of Structural Chemistry, University of Göttingen, 37077 Göttingen, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Schlicker
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, and the University of Göttingen, 37077 Göttingen, Germany.
authors.forename: Andrei
authors.initials: A
authors.lastname: Leonov
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, and the University of Göttingen, 37077 Göttingen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Zweckstetter
authors.affiliation: Department of Structural Chemistry, University of Göttingen, 37077 Göttingen, Germany.
authors.forename: George M
authors.initials: GM
authors.lastname: Sheldrick
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, and the University of Göttingen, 37077 Göttingen, Germany. Electronic address: sabe@nmr.mpibpc.mpg.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D000097624:Rabphilin-3A
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D008081:Liposomes; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D033921:Vesicular Transport Proteins; D000097624:Rabphilin-3A