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Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

Abstract

The neuronal protein synaptotagmin 1 functions as a Ca2+ sensor in exocytosis via two Ca2+-binding C2 domains. The very similar synaptotagmin 4, which includes all the predicted Ca2+-binding residues in the C2B domain but not in the C2A domain, is also thought to function as a neuronal Ca2+ sensor. Here we show that, unexpectedly, both C2 domains of fly synaptotagmin 4 exhibit Ca2+-dependent phospholipid binding, whereas neither C2 domain of rat synaptotagmin 4 binds Ca2+ or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca2+ ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. These results indicate that synaptotagmin 4 is a Ca2+ sensor in the fly but not in the rat, that the Ca2+-binding properties of C2 domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.

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Figure 1: Sequence comparison of the synaptotagmin 4 and 11 C2 domains.
Figure 2: Intrinsic Ca2+-binding properties of the synaptotagmin 4 and 11 C2 domains.
Figure 3: Ca2+-dependent phospholipid-binding properties of the synaptotagmin 4 and 11 C2 domains.
Figure 4: Crystal structure of the rat synaptotagmin 4 C2B domain.

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Acknowledgements

We thank A. Joachimiak and the staff of the Structural Biology Center beamlines 19BM and 19ID at the Advanced Photon Source for assistance in X-ray data collection. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the US Department of Energy, Office of Biological and Environmental Research, under contract no. W-31-109-ENG-38. This work was supported by US National Institutes of Health grant NS-40944 to J.R.

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Correspondence to Josep Rizo.

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Supplementary information

Supplementary Fig. 1

Superposition of the structures of the rat synaptotagmin 4 C2B-domain in the absence and presence of 100 mM Ca2+ (PDF 147 kb)

Supplementary Fig. 2

Slight Ca2+-induced shifts in the 1H-15N HSQC spectrum of the rat synaptotagmin 4 C2B-domain. (PDF 343 kb)

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Dai, H., Shin, OH., Machius, M. et al. Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4. Nat Struct Mol Biol 11, 844–849 (2004). https://doi.org/10.1038/nsmb817

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