Zheng Li, Chenavas Sylvie, Kieken Fabien, Trease Andrew, Brownell Sarah, Anbanandam Asokan, Sorgen Paul L, Spagnol Gaelle
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Biomolecular NMR Core Facility, University of Kansas, Lawrence, KS 66045, USA.
Biomolecules. 2020 Oct 17;10(10):1452. doi: 10.3390/biom10101452.
The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.
常染色体显性多效性疾病眼牙指发育异常（ODDD）由间隙连接蛋白Cx43的突变引起。在迄今确定的73种突变中，超过三分之一位于胞质环（Cx43CL）结构域。在此，我们确定了三种ODDD突变（M147T、R148Q和T154A）导致疾病的机制，这三种突变均位于Cx43CL预测的1-5-10钙调蛋白结合基序内。核磁共振（NMR）和圆二色性显示，这三种ODDD突变对Cx43CL形成α-螺旋结构以及结合钙调蛋白的能力几乎没有影响。显微镜检查和染料转移试验相结合发现，这些突变增加了Cx43的细胞内水平，而那些转运到质膜的突变并未形成功能性通道。NMR还确定钙调蛋白（CaM）可直接与Cx43CT结构域相互作用。参与CaM相互作用的Cx43CT残基与Pyk2和Src磷酸化的酪氨酸重叠。体外和细胞内数据提供了证据，表明CaM与Cx43CT相互作用的重要性可能在于限制Pyk2和Src磷酸化及其随后的下游效应。
