Vocke Farina, Weisschuh Nicole, Marino Valerio, Malfatti Silvia, Jacobson Samuel G, Reiff Charlotte M, Dell'Orco Daniele, Koch Karl-Wilhelm
Department of Neuroscience, Biochemistry Group, University of Oldenburg, Oldenburg, Germany.
Molecular Genetics Laboratory, Institute for Ophthalmic Research, University of Tübingen, Germany.
Hum Mol Genet. 2017 Jan 1;26(1):133-144. doi: 10.1093/hmg/ddw374.
Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.
黄斑营养不良会导致中心视力逐渐丧失，且表现出与年龄相关性黄斑变性相似的症状。对被诊断患有黄斑营养不良的患者进行基因筛查时，在GUCA1A基因中发现了一种新的突变，即c.526C>T替换，导致鸟苷酸环化酶激活蛋白1（GCAP1）中的氨基酸替换p.L176F。在三个呈现常染色体显性遗传模式的家族中发现了相同的变异。为了对L176F突变体进行全面的功能表征，我们表达并纯化了突变蛋白，并与野生型蛋白相比，测量了其激活特性、Ca2+/Mg2+结合以及Ca2+诱导的构象变化的关键参数。该突变体对游离Ca2+变化的敏感性较低，在生理Ca2+浓度下形成组成型活性形式，其激活速率比野生型显著更高（90倍对20倍），并且与其靶标鸟苷酸环化酶的相互作用具有更高的表观亲和力。然而，突变体的直接Ca2+结合与野生型几乎相似；Mg2+的结合具有更高的亲和力。我们进行了分子动力学模拟，以比较GCAP1的Ca2+饱和抑制状态与Mg2+结合的激活状态。当结合三个Ca2+或两个Mg2+时，L176F突变体表现出显著更低的灵活性，这可能构成了GCAP1功能改变的结构基础。
