TY - JOUR
T1 - The C2B domain is the primary Ca2 + sensor in DOC2B
T2 - A structural and functional analysis
AU - Giladi, Moshe
AU - Michaeli, Lirin
AU - Almagor, Lior
AU - Bar-On, Dana
AU - Buki, Tal
AU - Ashery, Uri
AU - Khananshvili, Daniel
AU - Hirsch, Joel A.
N1 - Funding Information:
We thank the staffs of ID-14-1, ID-23-1, and ID-23-2 at the European Synchrotron Radiation Facility for assistance with diffraction experimentation; the staff of X33 at DESY for assistance with SAXS experimentation; and Prof. Rimona Margalit and Dr. Ilia Rivkin for assistance with liposome preparation. This work was supported, in part, by Israel Science Foundation Grants 1211/07 and 730/11 and the German–Israeli Foundation Grant 1125-145.1/2010 to U.A. J.A.H. was funded by a German-Israeli Project Cooperation German Research Foundation grant. This work was partially funded by the Israeli Ministry of Health Grant 2010-3-6266 , the USA–Israel Binational Foundation Research Grant 2009-334 , and the Israel Science Foundation Grant 23/10 to D.K. M.G. was supported by a PhD fellowship from the Clore Scholars Program of the Clore Israel Foundation. The support of the Bernstein Foundation is highly appreciated.
PY - 2013/11/15
Y1 - 2013/11/15
N2 - DOC2B (double-C2 domain) protein is thought to be a high-affinity Ca2 + sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca2 +-binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure-function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca2 + with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca2 +. In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca2 + binding is stabilized, as reflected by the ∼ 10-fold slower rate of Ca2 + dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC50 of 400 nM while the C2A does not translocate at submicromolar Ca2 + concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an ∼ 2-fold lower EC50 and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca2 + sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.
AB - DOC2B (double-C2 domain) protein is thought to be a high-affinity Ca2 + sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca2 +-binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure-function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca2 + with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca2 +. In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca2 + binding is stabilized, as reflected by the ∼ 10-fold slower rate of Ca2 + dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC50 of 400 nM while the C2A does not translocate at submicromolar Ca2 + concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an ∼ 2-fold lower EC50 and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca2 + sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.
KW - DOC2B
KW - X-ray crystallography
KW - exocytosis
KW - small-angle X-ray scattering
KW - synaptic transmission
UR - http://www.scopus.com/inward/record.url?scp=84886728806&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2013.08.017
DO - 10.1016/j.jmb.2013.08.017
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C2 - 23994332
AN - SCOPUS:84886728806
SN - 0022-2836
VL - 425
SP - 4629
EP - 4641
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 22
ER -