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A conformational switch in the Piccolo C2A domain regulated by alternative splicing

Nature Structural & Molecular Biology volume 11, pages 45–53 (2004)Cite this article

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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Figure 1: Alternative splicing of the Piccolo C2A domain.
Figure 2: The Ca2+-binding properties of the short Piccolo C2A domain resemble those typical of C2 domains.
Figure 3: The short and long Piccolo C2A domain variants have similar structures in the presence of Ca2+ but not in the absence of Ca2+.
Figure 4: The short Piccolo C2A domain exhibits Ca2+-dependent binding to negatively charged phospholipids with much higher apparent Ca2+ affinity and lower salt sensitivity than the long form.
Figure 5: Three-dimensional structure of the Ca2+-free long Piccolo C2A domain.
Figure 6: Nature of the Ca2+-induced conformational change in the long Piccolo C2A domain.
Figure 7: Conformational change in the top loops required for Ca2+ binding to the long Piccolo C2A domain.

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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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Author notes

  1. Jesus Garcia

    Present address: RMN de Biomolecules, Parc Cientific de Barcelona, Barcelona, 08028, Spain

  2. Stefan H Gerber

    Present address: , Universitatsklinik Heidelberg Street, Heidelberg, M5T 1B3, Germany

  3. Shuzo Sugita

    Present address: Division of Cellular and Molecular Biology, Toronto Western Research Institute, 69120, Ontario, Canada

  4. Jesus Garcia and Stefan H Gerber: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Jesus Garcia & Josep Rizo

  2. Department of Molecular Genetics, Center for Basic Neuroscience, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Stefan H Gerber, Shuzo Sugita & Thomas C Südhof

  3. RMN de Biomolecules, Parc Cientific de Barcelona, Barcelona, 08028, Spain

    Stefan H Gerber & Shuzo Sugita

  4. Universitatsklinik Heidelberg Street, Heidelberg, M5T 1B3, Germany

    Jesus Garcia & Shuzo Sugita

  5. Division of Cellular and Molecular Biology, Toronto Western Research Institute, 69120, Ontario, Canada

    Jesus Garcia & Stefan H Gerber

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Correspondence to Jesus Garcia or Thomas C Südhof.

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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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