Colotti Gianni, Zamparelli Carlotta, Verzili Daniela, Mella Manuela, Loughrey Christopher M, Smith Godfrey L, Chiancone Emilia
Consiglio Nazionale delle Ricerche, Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, University of Rome La Sapienza, 00185 Rome, Italy.
Biochemistry. 2006 Oct 17;45(41):12519-29. doi: 10.1021/bi060416a.
Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca(2+) 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 Ca(2+) concentration as that of the wild type, whereas W105G requires a significantly higher Ca(2+) concentration. Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.
索辛蛋白是一种21.6 kDa的双结构域五聚体EF手型（PEF）蛋白，在与钙离子结合被激活后，会在一个很大程度上尚未明确的过程中与靶蛋白相互作用。两个具有生理功能的EF手型结构EF3和EF2并不属于一个结构对，但通过D螺旋相连。为了确定该螺旋是否在索辛蛋白激活过程中起作用，对D螺旋的两个残基进行了突变：位于EF3附近且参与相互作用网络的W105，以及位于EF2附近且面向溶剂的W99，将它们替换为甘氨酸。两种突变均未改变钙离子亲和力。研究了W105G和W99G突变体分别与膜联蛋白VII和心脏兰尼碱受体（RyR2）的相互作用，这两种相互作用分别需要索辛蛋白的N端和C端结构域。表面等离子体共振实验表明，膜联蛋白VII与W99G的结合在与野生型相同的钙离子浓度下发生，而W105G则需要显著更高的钙离子浓度。分离的心脏细胞的钙离子火花活性监测索辛蛋白与RyR2的相互作用，W105G对其无影响，但W99G和野生型对其降低程度相同。因此，通过破坏D螺旋相互作用网络来替换W105，会影响索辛蛋白在生理钙离子浓度下识别并与任一靶标相互作用的能力，而面向溶剂的W99的突变影响很小。D螺旋似乎放大了钙离子结合后在EF3处发生的局部结构变化，从而引发一种结构重排，使索辛蛋白能够与其分子靶标相互作用。鉴于D螺旋的保守性，相同的激活过程可能适用于其他PEF蛋白。
