The C2A-C2B Linker Defines the High Affinity Ca2+ Binding Mode of Rabphilin-3A

Pierre Montaville,Christine Schlicker,Andrei Leonov,Markus Zweckstetter,George M Sheldrick,Stefan Becker

doi:10.1074/jbc.m606746200

Pierre Montaville, Christine Schlicker + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m606746200

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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.

Highlights

Cesses preceding and/or following the vesicle fusion itself
Under low Ca2ϩ conditions, the C2B domain fragment crystallized in space group P21212 with one monomer in the asymmetric unit, whereas the presence of 200 mM Ca2ϩ in the reservoir resulted in crystals in space group P21 with two monomers in the asymmetric unit
The intense cross-peaks observed in the Ca2ϩ-free C2B domain spectrum, assigned to the acidic residues of the linker, were not seen in the liposomebound form of the C2B domain (Fig. 5, B and C). This suggests that the binding to the liposomes does not trigger the release of the linker. These observations strongly indicate that the structural features previously observed for the Ca2ϩbinding region (CBR) of the C2B domain in the presence of Ca2ϩ are maintained in the membrane-bound state

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cDNAs encoding fragments 371–510, 528 – 684, 519 – 684, and 371– 684 of rat rabphilin-3A were cloned into the expression vector pGEX2T (Amersham Biosciences). The model contains 149 residues for monomer A, 147 residues for monomer B, one phosphate group, two Ca2ϩ ions for each monomer, and a total of 77 water molecules. In both monomers, residues at the N and C termini are disordered (monomer A, residues 519 –523 and 679 – 684; and monomer B, residues 519 –524 and 678 – 684). The phosphate group forms a hydrogen bond with the backbone NH of Phe612 in monomer B and with Gly652 of a symmetry-related monomer B For both crystal structures, residues with completely or partially disordered side chains are listed in supplemental Tables 1 and 2. Backbone C-␣, C-␤, and nitrogen chemical shifts were obtained using standard

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RESULTS

DISCUSSION