Open AccessCalcium-Dependent Association of Calmodulin with the C-Terminal Domain of the Tetraspanin Protein Peripherin/rds
Author(s) -
Thomas Edrington,
Philip L. Yèagle,
Cheryl Gretzula,
Kathleen BoeszeBattaglia
Publication year - 2007
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/bi061999r
Subject(s) - calmodulin , transmembrane domain , biophysics , peripherin , chemistry , fusion protein , calcium binding protein , c terminus , transmembrane protein , plasma protein binding , calcium , biochemistry , biology , membrane , recombinant dna , receptor , amino acid , gene , organic chemistry
Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.