A conformational switch in the Piccolo C2A domain regulated by alternative splicing.
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 beta-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.
Garcia
Jesus
J
Department of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Gerber
Stefan H
SH
Sugita
Shuzo
S
Südhof
Thomas C
TC
Rizo
Josep
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; C405514:Pclo protein, rat; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D002118:Calcium