Montaville, PierrePierreMontavilleSchlicker, ChristineChristineSchlickerLeonov, AndreiAndreiLeonovZweckstetter, MarkusMarkusZweckstetterSheldrick, George M.George M.SheldrickBecker, StefanStefanBecker2018-11-072018-11-072007https://resolver.sub.uni-goettingen.de/purl?gro-2/51965The Ca2+ binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca2+ 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 Ca2+-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca2+ ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca2+ binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca2+-bound state of the C2B domain. In addition, this Ca2+ 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 Ca2+ binding mode not only shows how a C2 domain increases its intrinsic Ca2+ affinity, but also provides the structural base for an atypical protein-Ca2+-phospholipid binding mode of rabphilin-3A.journal_article10.1074/jbc.M60674620017166855000244482000084