The Center for Biomolecular Therapeutics (CBT), Department of Biochemistry and Molecular Biology University of Maryland School of Medicine, 108 N. Greene St, Baltimore, MD 21201, USA.
The Center for Biomolecular Therapeutics (CBT), Department of Biochemistry and Molecular Biology University of Maryland School of Medicine, 108 N. Greene St, Baltimore, MD 21201, USA; The Institute of Bioscience and Biotechnology Research (IBBR), 9600 Gudelsky Dr., Rockville, MD 20850, USA.
J Mol Biol. 2021 Nov 5;433(22):167272. doi: 10.1016/j.jmb.2021.167272. Epub 2021 Sep 27.
The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg-bound CaM (CaM). Upon titration of Ca into CaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca-binding domains, EF3 and EF4 (K = 60 ± 7 nM). As higher concentrations of free Ca were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (K = 1000 ± 160 nM). Thermodynamic and kinetic Ca binding studies showed that BP2 addition increased the Ca-binding affinity of CaM and slowed its Ca dissociation rates (k) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca concentrations (<100 nM) like those found at resting intracellular levels. As free Ca levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca-loaded CaM (CaM-BP2). Because this structural rearrangement observed for the CaM-BP2 complex occurs as intracellular free Ca concentrations approach those typical of a Ca-signaling event (K = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaM-STRA6.
钙调蛋白(CaM)与视黄醇摄取受体 STRA6 的相互作用涉及一个称为 BP2 的α-螺旋,该螺旋位于这种同源二聚体转运蛋白的细胞内侧(Chen 等人,2016 [1])。在没有 Ca 的情况下,NMR 数据表明,源自 BP2 的肽与 Mg 结合的 CaM(CaM)的 C 端结构域(C-结构域)结合。在将 Ca 滴定到 CaM-BP2 中时,观察到 NMR 化学位移扰动(CSP)发生在 C-结构域中的残基上,包括 EF 手型 Ca 结合域 EF3 和 EF4 中的残基(K = 60 ± 7 nM)。随着游离 Ca 浓度的升高,N-结构域(N-结构域)中的残基发生 CSP,包括 EF1 和 EF2 中的残基(K = 1000 ± 160 nM)。热力学和动力学 Ca 结合研究表明,BP2 的添加增加了 CaM 的 Ca 结合亲和力,并分别减慢了其在 C-和 N-结构域 EF 手型结构域中的 Ca 解离速率(k)。这些数据与 BP2 在低游离 Ca 浓度(<100 nM)下与 CaM 的 C-结构域结合的情况一致,这种情况类似于在静止的细胞内水平下发现的情况。当游离 Ca 水平接近 1000 nM 时,这是细胞内发生细胞内 Ca 信号事件时的典型情况,这里显示 BP2 与加载 Ca 的 CaM(CaM-BP2)的 N-和 C-结构域相互作用。由于观察到的 CaM-BP2 复合物的这种结构重排是在细胞内游离 Ca 浓度接近 Ca 信号事件的典型浓度时发生的(K = 1000 ± 160 nM),这种构象变化可能与全长 CaM-STRA6 的维生素 A 转运有关。