Structural assembly of the human Miro1/2 GTPases based on the crystal structure of the N-terminal GTPase domain

Abstract

PDB reference: Crystal structure of the human Miro1 N-terminal GTPase bound to GTP, 6D71 Abstract Dysfunction in mitochondrial dynamics is believed to contribute to a host of neurological disorders and has recently been implicated in cancer metastasis. The outer mitochondrial membrane adapter protein Miro functions in the regulation of mitochondrial mobility and degradation, however, the structural basis for its roles in mitochondrial regulation remain unknown. Here, we report a 1.7Å crystal structure of Nterminal GTPase domain (nGTPase) of human Miro1 bound unexpectedly to GTP, thereby revealing a non-catalytic configuration of the putative GTPase active site. We identify two conserved surfaces of the nGTPase, the "SELFYY" and "ITIP" motifs, that are potentially positioned to mediate dimerization or interaction with binding partners. Additionally, we report small angle X-ray scattering (SAXS) data obtained from the intact soluble HsMiro1 and its paralog HsMiro2. Taken together, the data allow modeling of a crescent-shaped assembly of the full-length soluble domain of HsMiro1/2.

Miro: A molecular switch at the center of mitochondrial regulation

“…For Miro2, these regions vary slightly in the C‐terminal GTPase domain, with Switch I corresponding to residues 446–455 and Switch II comprising residues 472–489 . Typically, in the GTP‐bound state, the surface loops are known as Switch I (G2) and Switch II (G3) and exist in close proximity to one another . However, the Rho‐conserved DxxG Switch II motif is absent from Miro1 .…”

“…While a full‐length structure of Miro has yet to be determined, there are two separate structural studies on Miro comprising a C‐terminal fragment (MiroS: EF1‐EF2‐cGTPase) and a recent structure of the nGTPase (Figure c). Previous work on MiroS indicated (a) the EF‐hands form a tightly packed interaction with the cGTPase and (b) Ca 2+ binding to the EF‐hands did not cause dramatic structural changes.…”

“…Recently, the structure of the isolated nGTPase was determined using X‐ray crystallography . Surprisingly, despite the absence of nucleotide during protein purification and crystallization, the nGTPase crystallized with GTP bound to the active site . This suggests that the nGTPase alone may not be sufficient for GTP hydrolysis.…”

“…However, size‐exclusion chromatography and small‐angle X‐ray scattering indicated that these constructs were monomers in solution, allowing the authors to conclude that Miro does not dimerize in solution. Finally, recent small‐angle X‐ray scattering studies suggest that the nGTPase may be loosely tethered to the MiroS . This suggests that the nGTPase may exist in multiple conformations or that the nGTPase is capable of interacting with MiroS.…”

“…This will involve mutations that alter the GTP state, phosphorylation sites, and calcium binding. To help the reader, we have constructed an in silico model of Miro based on superimposing Miro's nGTPase with the EF1‐EF2‐cGTPase structure (Figure c).…”