Wright Katharine M, Wu Kuanlin, Babatunde Omotolani, Du Haijuan, Massiah Michael A
Department of Chemistry, George Washington University, Washington, D.C., United States of America.
PLoS One. 2014 Sep 12;9(9):e107537. doi: 10.1371/journal.pone.0107537. eCollection 2014.
MID1 catalyzes the ubiquitination of the protein alpha4 and the catalytic subunit of protein phosphatase 2A. Mutations within the MID1 Bbox1 domain are associated with X-linked Opitz G syndrome (XLOS). Our functional assays have shown that mutations of Ala130 to Val or Thr, Cys142 to Ser and Cys145 to Thr completely disrupt the polyubiquitination of alpha4. Using NMR spectroscopy, we characterize the effect of these mutations on the tertiary structure of the Bbox1 domain by itself and in tandem with the Bbox2 domain. The mutation of either Cys142 or Cys145, each of which is involved in coordinating one of the two zinc ions, results in the collapse of signal dispersion in the HSQC spectrum of the Bbox1 domain indicating that the mutant protein structure is unfolded. Each mutation caused the coordination of both zinc ions, which are ∼ 13 Å apart, to be lost. Although Ala130 is not involved in the coordination of a zinc ion, the Ala130Thr mutant Bbox1 domain yields a poorly dispersed HSQC spectrum similar to those of the Cys142Ser and Cys145Thr mutants. Interestingly, neither cysteine mutation affects the structure of the adjacent Bbox2 domain when the two Bbox domains are engineered in their native tandem Bbox1-Bbox2 protein construct. Dynamic light scattering measurements suggest that the mutant Bbox1 domain has an increased propensity to form aggregates compared to the wild type Bbox1 domain. These studies provide insight into the mechanism by which mutations observed in XLOS affect the structure and function of the MID1 Bbox1 domain.
MID1催化蛋白质α4和蛋白磷酸酶2A催化亚基的泛素化。MID1 Bbox1结构域内的突变与X连锁Opitz G综合征（XLOS）相关。我们的功能分析表明，Ala130突变为Val或Thr、Cys142突变为Ser以及Cys145突变为Thr的突变完全破坏了α4的多聚泛素化。我们使用核磁共振光谱法，单独以及与Bbox2结构域串联，表征了这些突变对Bbox1结构域三级结构的影响。参与配位两个锌离子之一的Cys142或Cys145的突变，导致Bbox1结构域的HSQC谱中信号分散性丧失，表明突变蛋白结构展开。每个突变都导致相距约13 Å的两个锌离子的配位丢失。尽管Ala130不参与锌离子的配位，但Ala130Thr突变的Bbox1结构域产生的HSQC谱分散性较差，类似于Cys142Ser和Cys145Thr突变体的谱。有趣的是，当两个Bbox结构域构建在其天然串联的Bbox1 - Bbox2蛋白构建体中时，两个半胱氨酸突变均不影响相邻Bbox2结构域的结构。动态光散射测量表明，与野生型Bbox1结构域相比，突变的Bbox1结构域形成聚集体的倾向增加。这些研究深入了解了XLOS中观察到的突变影响MID1 Bbox1结构域结构和功能的机制。
