Nalefski E A, Falke J J
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Biochemistry. 1998 Dec 22;37(51):17642-50. doi: 10.1021/bi982372e.
Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.
C2结构域与靶膜的对接是由多个Ca2+离子与嵌入在三个其他可变的Ca2+结合环内的保守残基阵列结合引发的。我们通过在广泛分布于cPLA2的Ca2+激活的C2结构域表面的16个不同位置进行单个半胱氨酸取代，确定了该结构域的膜对接表面，在每种情况下都为荧光素探针生成一个独特的附着位点。荧光素标记的半胱氨酸的环境敏感性使得能够识别一个受Ca2+结合和膜对接干扰的局部区域。Ca2+与该结构域的结合改变了包含Ca2+结合环的区域中六个荧光素的发射强度，表明Ca2+触发的环境变化局限于该区域。同样，膜对接增加了Ca2+结合环区域内六个荧光素的质子化，表明这三个环也直接参与膜对接。此外，碘化物猝灭测量表明，膜对接使三个荧光素标记位置，即苯丙氨酸35、天冬酰胺64和酪氨酸96，免受与水相碘离子的碰撞。这些被隔离的残基位于已确定的膜对接区域内，在三个Ca2+结合环中的每一个环中各有一个。最后，仅半胱氨酸取代就足以仅在苯丙氨酸35和酪氨酸96位置显著降低膜亲和力，突出了这两个环残基在膜对接中的重要性。总之，结果表明C2结构域的膜对接表面定位于协同结合一对Ca2+离子的同一表面，并且三个Ca2+结合环本身提供了大部分或全部的膜接触。这些以及其他结果进一步支持了C2结构域膜特异性的一般模型，其中可变的Ca2+结合环在由多个Ca2+离子稳定的蛋白质-膜界面处提供头部基团识别。
