Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA.
J Mol Biol. 2011 Apr 8;407(4):505-20. doi: 10.1016/j.jmb.2011.01.048. Epub 2011 Feb 4.
Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains-RING (really interesting new gene), Bbox1, and Bbox2-that have similar ββα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function.
人 MID1（中线-1）是一种微管相关蛋白，据推测它将蛋白磷酸酶 2A 的催化亚基靶向降解。它与α4 结合，然后招募蛋白磷酸酶 2A 的催化亚基。作为 TRIM（三部分基序）家族的一员，MID1 具有三个连续的锌结合结构域-RING（真正有趣的新基因）、Bbox1 和 Bbox2-它们具有相似的 ββα 折叠。在这里，我们分别描述了这些结构域以及串联结构域的体外特性。我们观察到 RING 结构域表现出比 Bbox 结构域更高的泛素（Ub）E3 连接酶活性。RING-Bbox1 和 RING-Bbox1-Bbox2 串联结构域的自聚泛素化产物的量明显大于单个结构域的量。然而，串联的 Bbox1-Bbox 结构域没有观察到多泛素化产物。使用 Ub 突变体，我们观察到这些 MID1 结构域构建体通过 Ub 的 Lys63 促进 Ub 链延伸。此外，我们观察到高分子量蛋白产物主要是由于 RING-Bbox1 和 RING-Bbox1-Bbox2 构建体的 Bbox1 结构域上一个位点（Lys154）的多泛素化。我们观察到 MID1 E3 结构域可以与多种 E2 连接酶相互作用。最后，从α4 的 C 末端衍生的与 Bbox1 紧密结合的 45 个氨基酸肽在该测定中被观察到单泛素化，并且似乎下调了形成的多泛素化产物的量。这些研究揭示了 MID1 E3 连接酶活性，并展示了其三个锌结合结构域如何有助于 MID1 的整体功能。
