Browsing by Author "Bill, Roslyn M"

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    Simultaneous binding of the N- and C-terminal cytoplasmic domains of aquaporin 4 to calmodulin
    (Elsevier, 2022-01) Ishida, Hiroaki; Vogel, Hans J; Conner, Alex C; Kitchen, Philip; Bill, Roslyn M; MacDonald, Justin A
    Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; Kd1: 3.6 μM, Kd2: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 μM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data support a unique adapter protein binding mode for CaM with AQP4.