The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca2+-dependent interaction with annexin VII and the cardiac ryanodine receptor
The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca2+-dependent interaction with annexin VII and the cardiac ryanodine receptor
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DOI：
10.1021/bi060416a
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发表时间：
2006-10-17
期刊：
影响因子：
2.9
通讯作者：
Chiancone, Emilia
中科院分区：
文献类型：
--
作者：
Colotti, Gianni;Zamparelli, Carlotta;Chiancone, Emilia
Sorcin是一种分子量为21.6 kDa的双结构域penta-EF-hand（PEF）蛋白，当被Ca 2+激活时，其与靶蛋白的相互作用过程在很大程度上是未知的。两个生理EF-手EF 3和EF 2不属于结构对，但通过D螺旋连接。为了确定该螺旋是否有助于sorcin活化，突变了两个D螺旋残基：W105，位于EF 3附近并参与相互作用网络，W 99，位于EF 2附近并面对溶剂，用甘氨酸取代。这两种突变都不会改变钙的亲和力。研究了W105 G和W 99 G突变体与膜联蛋白VII和心脏兰尼碱受体（RyR 2）的相互作用，其分别需要sorcin N-末端和C-末端结构域。表面等离子体共振实验表明，结合膜联蛋白VII W 99 G发生在相同的Ca 2+浓度的野生型，而W105 G需要一个显着更高的Ca 2+浓度。分离的心脏细胞的Ca 2+火花活性监测sorcin-RyR 2相互作用，并且不被W105 G改变，但被W 99 G和野生型同等地降低。因此，取代W105，通过破坏网络的D螺旋相互作用，影响的能力sorcin识别和相互作用的目标在生理Ca 2+浓度，而突变的溶剂面临W 99几乎没有影响。D螺旋似乎放大了Ca 2+结合后EF 3处发生的局部结构变化，从而触发了使sorcin与其分子靶点相互作用的结构重排。鉴于D螺旋保守性，相同的活化过程可适用于其他PEF蛋白。
Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca2+ binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N- terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca2+ concentration as that of the wild type, whereas W105G requires a significantly higher Ca2+ concentration. Ca2+ spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca2+ concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca2+ binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.