XLOS-observed mutations of MID1 Bbox1 domain cause domain unfolding

Abstract

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.

Figure 1. Structure of MID1 Bbox1 and Bbox2 domains.

A. Ribbon representation of the tertiary structure of the MID1 Bbox1 domain. The Bbox1 domain coordinates two zinc ions and adopts a ββα-RING fold. Amino acid residues coordinating the zinc ions are labeled. Spheres (magenta) represent the two zinc ions. B. Ribbon representation of the MID1 Bbox1-Bbox2 domains in tandem. Each Bbox domain adopts a similar ββα-RING fold. The structure of the Bbox1 domain is shown with a similar orientation as the structure of the Bbox1 domain alone in A. The side chain methyl group of Ala130 is shown as spheres.

Figure 2. ¹H-¹⁵N HSQC spectra of the Cys142Ser mutant Bbox1 domain.

A. Superposition of the HSQC spectrum of wild type MID1 Bbox1 (black) on that of the Cys142Ser mutant Bbox1 domain (red). B. Superposition of the HSQC spectrum of Cys142Ser mutant Bbox1 domain in the absence (red) and presence (green) of EDTA. C. Superposed HSQC spectra of the wild type Bbox1 domain in the presence of EDTA (blue) and the Cys142Ser Bbox1 domain (red).

Figure 3. ¹H-¹⁵N HSQC spectra of the Cys145Thr mutant Bbox1 domain.

A. Superposed spectrum of wild type LB1B2 (black) on that of the Cys145Thr mutant Bbox1 domain (red). Cross peaks belonging to amino acids of the Bbox1 domain in the wild type folded LB1B2 construct are labeled. Cross peaks for the Cys145Thr Bbox1 domain, which is unfolded, are not readily identifiable. The additional poorly dispersed cross peaks in the center of Figure 3B, compared to the number of poorly dispersed peaks for just the Bbox1 domain shown in Figure 2A, belong to unstructured residues 71–115. B. Superposition of the HSQC spectra of Cys145Thr LB1B2 protein (red) and the isolated Bbox2 domain (green). The near exact overlap of signals indicates that the Bbox2 domain within the mutant tandem construct is structured.

Figure 4. ¹H-¹⁵N HSQC spectra of the Ala130Thr mutant Bbox1 domain.

A. Superposed HSQC spectra of wild type Bbox1 domain (black) and the Ala130Thr mutant Bbox1 domain (blue). B. Superposition of the HSQC spectra of Ala130Thr (blue) and Cys142Ser (red) mutant Bbox1 domains.

Figure 5. Structural and functional effects of the Ala130Ser mutant Bbox1 domain.

A. Superposition of the HSQC spectra of Ala130Ser (green) and the Ala130Thr Bbox1 domains (blue). B. Western blot showing ubiquitination of alpha4 by the wild type RING-Bbox1-Bbox2 (RB1B2) domain construct and RB1B2 harboring the Ala130Ser mutation. Lane 2 shows the result of a control reaction in which the E3 ligase was omitted. Two strong bands indicating mono- and di-ubiquitinated alpha4 as well as a smearing pattern indicative of polyubiquitinated alpha4 was observed for the Ala130Ser Bbox1 domain.

Figure 6. ¹H-¹⁵N-HSQC spectra of the Ala130Thr mutant Bbox1 domain in tandem LB1B2.

A. Superposition of the spectrum of Ala130Thr LB1B2 (cyan) on that of wild type LB1B2 (black). Cross peaks for amino acids in the wild type Bbox1 domain are labeled on the wild type LB1B2 spectrum. B. Superposition of the spectra of the Ala130Thr domain (cyan) and Cys145Thr (red) LB1B2 protein (red).

Figure 7. Model of the A130 mutants.

A. Ribbon drawing of MID1 Bbox1 domain with sphere representation for the side chain atoms of Ala130, Cys119 and Cys142. Top right: a close-up view of these three residues. Bottom right: a top-down view with the molecule rotated as indicated to highlight the position of the methyl group of Ala130 relative to the methylene atoms of the two cysteine residues. B. Views from the same orientation as in A showing a model in which a threonine replaces Ala130. The space-filling model reveals that the methyl group of the threonine would clash with the methylene atoms of Cys119 and Cys142. C. Close-up views of the position of the side chain locations in a model of the Ala130Ser mutation. Shown is a large cavity and that could accommodate each of three possible rotameric states of the serine side chain. As a result, the serine hydroxyl group could be involved in different hydrogen-bonds. The figures were generated with Pymol.