Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.
College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.
Biochim Biophys Acta Biomembr. 2022 Feb 1;1864(2):183837. doi: 10.1016/j.bbamem.2021.183837. Epub 2021 Dec 7.
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 Ca-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional H, N-HSQC NMR with N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a K of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; K: 3.6 μM, K: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a K 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.
水通道蛋白 4（AQP4）是一种水转运的跨膜通道蛋白，在维持细胞内水稳态方面具有重要的调节作用。其他几种 AQP 蛋白表现出钙调蛋白（CaM）结合特性，最近 CaM 被牵连到 AQP4 的细胞表面定位。本研究的目的是详细评估 AQP4 的 CaM 结合特性。AQP4 的检查显示细胞质 N-和 C-末端区域分别存在两个假定的 CaM 结合结构域（CBD）。使用荧光光谱法、等温滴定量热法和二维 H、N-HSQC NMR 与 N 标记的 CaM 评估 AQP4 CBD 肽的 Ca 依赖性 CaM 结合特性。AQP4 的 N-末端 CBD 主要与 CaM 的 N- lobe 相互作用，结合比为 1:1，K 值为 3.4 μM。C-末端 AQP4 肽与 CaM 的 C-和 N- lobe 相互作用（结合比为 2:1；K 值分别为 3.6 μM 和 113.6 μM）。含有整个细胞质 C-末端序列的重组 AQP4 蛋白结构域（recAQP4）以 1:1 结合模式与 CaM 结合，K 值为 6.1 μM。可以生成三元桥接复合物，CaM 的 N-和 C- lobe 同时与 N-和 C-末端 CBD 肽相互作用。这些数据支持 CaM 与 AQP4 之间存在独特的衔接蛋白结合模式。
