Amann K J, Renley B A, Ervasti J M
Graduate Program in Cellular and Molecular Biology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J Biol Chem. 1998 Oct 23;273(43):28419-23. doi: 10.1074/jbc.273.43.28419.
The dystrophin rod domain is composed of 24 spectrin-like repeats and was thought to act mainly as a flexible spacer between the amino-terminal actin binding domain and carboxyl-terminal membrane-associated domains. We previously demonstrated that a fragment of the dystrophin rod domain also binds F-actin. However, the nature and extent of rod domain association with F-actin is presently unclear. To begin addressing these questions, we characterized two recombinant proteins representing adjacent regions of the dystrophin rod. DYS1416 (amino acids 1416-1880) bound F-actin with a Kd of 14.2 +/- 5.2 microM and a stoichiometry of 1 mol:mol of actin. However, DYS1030 (amino acids 1030-1494) failed to bind F-actin, suggesting that not all rod domain repeats are capable of binding F-actin. Interestingly, DYS1416 corresponds to a unique region of the dystrophin rod rich in basic amino acids, whereas DYS1030 is composed mainly of acidic repeats. This observation suggested that DYS1416 may interact with acidic actin filaments through an electrostatic interaction. Supporting this hypothesis, actin binding by DYS1416 was dramatically inhibited by increasing ionic strength. We suggest that electrostatic interactions between basic spectrin-like repeats and actin filaments may contribute to the actin binding activity of other members of the actin cross-linking protein family.
肌营养不良蛋白杆状结构域由24个血影蛋白样重复序列组成，被认为主要作为氨基末端肌动蛋白结合结构域和羧基末端膜相关结构域之间的柔性间隔区发挥作用。我们之前证明了肌营养不良蛋白杆状结构域的一个片段也能结合F-肌动蛋白。然而，目前尚不清楚杆状结构域与F-肌动蛋白结合的性质和程度。为了开始解决这些问题，我们对代表肌营养不良蛋白杆相邻区域的两种重组蛋白进行了表征。DYS1416（氨基酸1416 - 1880）以14.2 +/- 5.2 microM的解离常数（Kd）和1摩尔肌动蛋白对应1摩尔的化学计量比结合F-肌动蛋白。然而，DYS1030（氨基酸1030 - 1494）未能结合F-肌动蛋白，这表明并非所有杆状结构域重复序列都能结合F-肌动蛋白。有趣的是，DYS1416对应于肌营养不良蛋白杆中富含碱性氨基酸的一个独特区域，而DYS1030主要由酸性重复序列组成。这一观察结果表明，DYS1416可能通过静电相互作用与酸性肌动蛋白丝相互作用。支持这一假设的是，增加离子强度会显著抑制DYS1416与肌动蛋白的结合。我们认为碱性血影蛋白样重复序列与肌动蛋白丝之间的静电相互作用可能有助于肌动蛋白交联蛋白家族其他成员的肌动蛋白结合活性。
