Peshenko Igor V, Dizhoor Alexander M
Hafter Research Laboratories, Pennsylvania College of Optometry, Elkins Park, PA 19027, USA.
J Biol Chem. 2007 Jul 27;282(30):21645-52. doi: 10.1074/jbc.M702368200. Epub 2007 Jun 1.
Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.
鸟苷酸环化酶激活蛋白1（GCAP-1）是一种Ca(2+)/Mg(2+)传感蛋白，在光照下加速视网膜鸟苷酸环化酶（RetGC），在黑暗中使其减速，以无阳离子形式存在时无活性。在模拟光适应的条件下，EF-手型结构2和3中Mg(2+)的结合对于RetGC激活至关重要。EF-手型结构2中Mg(2+)的结合影响相邻的非金属结合结构域EF-手型结构1的构象，与无金属的EF-手型结构2相比，增加了GCAP-1对RetGC的亲和力近40倍。EF-手型结构3中Mg(2+)的结合使GCAP-1对RetGC的亲和力增加5倍，其对RetGC的最大刺激增加2倍。EF-手型结构4中Mg(2+)的结合既不影响GCAP-1对RetGC的亲和力，也不影响RetGC的激活。EF-手型结构4中Ca(2+)结合的失活足以使GCAP-1成为RetGC的组成型激活剂，而EF-手型结构3在Ca(2+)依赖性RetGC减速中的作用可能是通过相邻的EF-手型结构4。EF-手型结构2中Ca(2+)结合的失活影响Ca(2+)对RetGC抑制的协同性，但不能阻止抑制作用。我们得出结论：1）EF-手型结构2和3而非EF-手型结构4中Mg(2+)的结合对于GCAP-1在光照下激活RetGC的能力至关重要；2）EF-手型结构3尤其是EF-手型结构2中Mg(2+)或Ca(2+)的结合是与环化酶高亲和力相互作用所必需的，并影响相邻的EF-手型结构1的构象，EF-手型结构1是靶向RetGC所需的结构域；3）RetGC抑制可能主要由EF-手型结构4中Ca(2+)的结合引起。
