Li Yi, Wright Nathan T, Bloch Robert J
Program in Biochemistry and Molecular Biology, University of Maryland Baltimore, Baltimore, Maryland, USA.
Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia, USA.
J Biol Chem. 2025 Mar;301(3):108216. doi: 10.1016/j.jbc.2025.108216. Epub 2025 Jan 23.
Sarcoplasmic/endoplasmic reticulum Ca-ATPase1 (SERCA1) is responsible for the clearance of cytosolic Ca in skeletal muscle. Due to its vital importance in regulating Ca homeostasis, the regulation of SERCA1 has been intensively studied. Small ankyrin 1 (sAnk1, Ank1.5), a 17 kDa muscle-specific isoform of ANK1, binds to SERCA1 directly via both its transmembrane and cytoplasmic domains and inhibits SERCA1's ATPase activity. Here, we characterize the interaction between the cytoplasmic domain of sAnk1 (sAnk1(29-155)) and SERCA1. The binding affinity for sAnk1 (29-155) to SERCA1 was 444 nM by blot overlay, about 7-fold weaker than the binding of sAnk1(29-155) to obscurin, a giant protein of the muscle cytoskeleton. Site-directed mutagenesis identified K38, H39, and H41, in the juxtamembrane region, as residues likely to mediate binding to SERCA1. These residues are not required for obscurin binding. Residues R64-K73, which do contribute to obscurin binding, are also required for binding to SERCA1, but only the hydrophobic residues in this sequence are required, not the positively charged residues necessary for obscurin binding. Circular dichroism analysis of sAnk1(29-155) indicates that most mutants show significant structural changes, with the exception of those containing alanines in place of K38, H39 and H41. Although the cytoplasmic domain of sAnk1 does not inhibit SERCA1's Ca-ATPase activity, with or without mutations in the juxtamembrane sequence, the inhibitory activity of full-length sAnk1 requires the WT juxtamembrane sequence. We used these data to model sAnk1 and the sAnk1-SERCA1 complex. Our results suggest that, in addition to its transmembrane domain, sAnk1 uses its juxtamembrane sequence and perhaps part of its obscurin binding site to bind to SERCA1, and that this binding contributes to their robust association in situ, as well as regulation of SERCA1's activity.
肌浆网/内质网Ca-ATP酶1（SERCA1）负责清除骨骼肌胞质中的Ca。由于其在调节Ca稳态中至关重要，因此对SERCA1的调节已进行了深入研究。小锚蛋白1（sAnk1，Ank1.5）是ANK1的一种17 kDa肌肉特异性异构体，通过其跨膜结构域和细胞质结构域直接与SERCA1结合，并抑制SERCA1的ATP酶活性。在此，我们对sAnk1的细胞质结构域（sAnk1(29-155)）与SERCA1之间的相互作用进行了表征。通过印迹覆盖法测定，sAnk1(29-155)与SERCA1的结合亲和力为444 nM，比sAnk1(29-155)与 obscurin（一种肌肉细胞骨架的巨型蛋白）的结合弱约7倍。定点诱变确定，近膜区域的K38、H39和H41是可能介导与SERCA1结合的残基。这些残基对于与obscurin结合并非必需。对obscurin结合有贡献的R64-K73残基对于与SERCA1结合也是必需的，但该序列中仅疏水残基是必需的，而不是obscurin结合所需的带正电荷残基。对sAnk1(29-155)的圆二色性分析表明，除了那些用丙氨酸取代K38、H39和H41的突变体之外，大多数突变体都显示出显著的结构变化。尽管sAnk1的细胞质结构域不抑制SERCA1的Ca-ATP酶活性，无论近膜序列是否存在突变，但全长sAnk1的抑制活性需要野生型近膜序列。我们利用这些数据构建了sAnk1和sAnk1-SERCA1复合物的模型。我们的结果表明，除了其跨膜结构域之外，sAnk1还利用其近膜序列以及可能其obscurin结合位点的一部分来与SERCA1结合，并且这种结合有助于它们在原位的稳固结合以及对SERCA1活性的调节。
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