Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

Abstract

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

Figure 1. Potential two AAA+ modules of mysterin.

(A) Predicted secondary structures surrounding Walker A and B motifs of mysterin resemble those of typical AAA+ ATPases, which contains indicated secondary structures (α and β refer to α helix and β strand, respectively) and elements: Walker A (x indicates any amino acid) and B (h indicates hydrophobic residue) motifs, pore region, arginine finger, and sensor 1 and 2 regions. Definitions of the structural elements are based on Ref. . (B) Homology model of AAA+ modules of mouse mysterin based on a crystal structure of Thermus thermophilus ClpB (PDB ID: 1QVR).

Figure 2. ATPase and ATP-binding activities of the first and the second AAA+ ATPase modules.

(A) Affinity-purified N-terminally GST-fused first and second AAA+ modules were incubated with 5 mM ATP. The free phosphate released by ATP hydrolysis was measured by malachite green assay. Error bars represent the standard errors from the three independent experiments. (B) Affinity-purified N-terminally GST-fused first and second AAA+ modules were incubated with ATP agarose beads, and the bound protein was detected by Western blotting using anti-GST antibody. Full-length blots are presented in Supplementary Fig. 4A.

Figure 3. Electron micrographs of negative stained samples demonstrate ring-shaped oligomer of purified mysterin.

(A) C-terminally 3 × FLAG-tagged mysterin was purified using anti-FLAG affinity agarose beads. The indicated volume of purified protein sample was applied to a 3%–10% gradient SDS gel and was silver stained. The arrowhead indicates the 591-kDa mysterin. (B) Electron micrographs of negative stained samples demonstrate ring-shaped particles.

Figure 4. ATP hydrolysis of the second AAA+ module destabilizes mysterin homo-oligomers.

(A) Schematic representation of the biochemical assay used for detecting mysterin homo-oligomer. (B) The complex of mysterin-3 × FLAG and mysterin-Myc was detected as schematically represented in (A). Full-length blots are presented in Supplementary Fig. 4B. (C) Schematic representation of AAA+ combination mutants of mysterin. A1, A2, B1, and B2 indicate the first and second Walker A motifs and the first and second Walker B motifs, respectively. (D) The A2B2 and B1B2 mutants exhibited significantly stronger homo-oligomer formation than did wild-type mysterin. Full-length blots are presented in Supplementary Fig. 4C. (E) The B2 single mutant solely showed enhanced homo-oligomer formation. Full-length blots are presented in Supplementary Fig. 4D. (F) The A2 single mutant solely showed enhanced homo-oligomer formation. Full-length blots are presented in Supplementary Fig. 4E.

Figure 5. The mysterin oligomer is differently regulated by nucleotide binding to the first AAA+ module and ATP hydrolysis on the second AAA+ module.

(A) A1A2 and A1B2 showed regular oligomer formation strength. Full-length blots are presented in Supplementary Fig. 4F. (B) Schematic representation of the results obtained by the oligomer formation assay. The four mutants containing mutations in the second module showed strong oligomer formation (+++), whereas A1A2, A1B2 and A1B1A2B2 showed regular assembly strength (+). (C) Model of the relationships between mysterin oligomerization and AAA+ activities.