PDBsum entry 1k5w

Endocytosis/exocytosis

PDB id: 1k5w

Contents

Protein chain

148 a.a. *

Metals

_CA ×2

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Three-Dimensional structure of the synaptotagmin 1 c2b-Domain: synaptotagmin 1 as a phospholipid binding machine.

Authors

I.Fernandez, D.Araç, J.Ubach, S.H.Gerber, O.Shin, Y.Gao, R.G.Anderson, T.C.Südhof, J.Rizo.

Ref.

Neuron, 2001, 32, 1057-1069. [DOI no: 10.1016/S0896-6273(01)00548-7]

PubMed id

11754837

Abstract

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.

Figure 1.
Figure 1. Three-Dimensional Structure of the Synaptotagmin 1 C2B-Domain. (A) and (B) show superpositions of the 20 structures with the lowest NOE energies in two different orientations. (C) and (D) are ribbon diagrams of the structure of the C2B-domain in the same orientations. Strands are labeled from 1 to 8, helices are labeled HA and HB, and the Ca2+ ions (orange spheres) are labeled Ca1 and Ca2. Note that the loop connecting strands 5 and 6 forms a very short alpha-helix in some C2-domains but such helix is not observed in the structure of the synaptotagmin 1 C2B-domain. The figure was generated with the programs InsightII (MSI, San Diego, California) and Molscript (Kraulis, 1991).

Figure 7.
Figure 7. The Synaptotagmin 1 C[2]B-Domain Binds PS-Containing Vesicles in a Ca^2+-Dependent Manner(A) and (B) show FRET measurements from the C[2]A-domain (A) and C[2]B-domain (B) to phospholipid vesicles containing 10% dansyl-PE, 25% PS, and 65% PC in the presence of 0.5 mM EDTA (red traces) or 0.2 mM Ca^2+ (black traces). The protein concentration was 1 Ž§¼M and the lipid concentration was 0.022 mg/ml. In (B), the green trace was acquired with wild-type C[2]B-domain in 1 mM Mg^2+ and the blue trace with the D309N mutant C[2]B-domain in the presence of 0.2 mM Ca^2+. A spectrum acquired under identical conditions but without protein was subtracted for each data set. (C) and (D) show Ca^2+ dependence of phospholipid binding measured by FRET for the C[2]A-domain (C) and the C[2]B-domain (D). The relative change in FRET measured as in (A) and (B) is represented as a function of the Ca^2+ concentration for the C[2]A-domain (C) and the C[2]B-domain (D). The data represent an average of three measurements which yielded an apparent Ca^2+ affinity and Hill coefficient of 54 Ž§¼M Ca^2+ and 1.3, respectively, for the C[2]A-domain, and 48 Ž§¼M Ca^2+ and 1.6, respectively, for the C[2]B-domain. (E) shows Ca^2+-dependent phospholipid binding to the C[2]A-domain and the C[2]B-domain monitored by centrifugation. Samples containing purified GST-C[2]A-domain or GST-C[2]B-domain were incubated with liposomes (PS/PC 25:75) and various Ca^2+ concentrations as indicated. The samples were centrifuged and, after washing the precipitated liposomes, bound protein was analyzed by SDS-PAGE and Coomassie Blue staining. (F) and (G) show Ca^2+-dependent phospholipid binding to immobilized GST-C[2]A-domain (F) and GST-C[2]B-domain (G). GST fusion proteins reattached to glutathione-agarose after purification in solution were incubated with ^3H-labeled liposomes (PS/PC 30:70) at various Ca^2+ concentrations and after washing the resin, the bound lipids were measured by scintillation counting. Each graph shows a representative experiment performed in triplicate. The average apparent Ca^2+ affinity and Hill coefficient obtained from four independent experiments was 13 Ž§¼M Ca^2+ and 3.6, respectively, for the C[2]A-domain, and 15 Ž§¼M Ca^2+ and 1.7, respectively, for the C[2]B-domain. Some error bars are not visible because of their small size.

The above figures are reprinted by permission from Cell Press: Neuron (2001, 32, 1057-1069) copyright 2001.