Abstract
C2 domains are widespread Ca2+-binding modules. The active zone protein Piccolo (also known as Aczonin) contains an unusual C2A domain that exhibits a low affinity for Ca2+, a Ca2+-induced conformational change and Ca2+-dependent dimerization. We show here that removal of a nine-residue sequence by alternative splicing increases the Ca2+ affinity, abolishes the conformational change and abrogates dimerization of the Piccolo C2A domain. The NMR structure of the Ca2+-free long variant provides a structural basis for these different properties of the two splice forms, showing that the nine-residue sequence forms a β-strand otherwise occupied by a nonspliced sequence. Consequently, Ca2+-binding to the long Piccolo C2A domain requires a marked rearrangement of secondary structure that cannot occur for the short variant. These results reveal a novel mechanism of action of C2 domains and uncover a structural principle that may underlie the alteration of protein function by short alternatively spliced sequences.
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Acknowledgements
We thank I. Leznicki, A. Roth, and E. Borowicz for technical assistance, I. Fernandez for initial studies on the Ca2+ affinity of the Piccolo C2A domain and D. Myers for assistance with analytical ultracentrifugation. This study was supported by grants from the Welch Foundation (I-1304, J.R.), the Perot Family Foundation (T.C.S.), US National Institutes of Health grant NS40944 (J.R. and T.C.S.) and a fellowship from the Deutsche Forschungsgemeinschaft (S.H.G).
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Garcia, J., Gerber, S., Sugita, S. et al. A conformational switch in the Piccolo C2A domain regulated by alternative splicing. Nat Struct Mol Biol 11, 45–53 (2004). https://doi.org/10.1038/nsmb707
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DOI: https://doi.org/10.1038/nsmb707
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