Structure and Assembly of Human Miro1/2 GTPases and their Putative Effectors

Smith, Kyle P

doi:https://doi.org/10.21985/n2-j7dv-ys49

Work

Structure and Assembly of Human Miro1/2 GTPases and their Putative Effectors

Public

Download PDF

Download All Files (.zip)

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 proteins Miro1 and Miro2 play a role in the regulation of mitochondria. Each paralog comprises two GTPase domains flanking two centrally located calcium/magnesium-binding helix-loop-helix (EF-hand) domains. However, the biochemical and structural mechanisms by which Miro influences mitochondria are unknown. Here we report the crystal structure of the N-terminal GTPase domain (nGTPase) of human Miro1 and propose evidence of both monomer and dimerization of the assembly of the full-length soluble domain of human Miro1 and Miro2. The nGTPase structure at 1.7 Å resolution shows an irregular active site, including backbone coordination of the magnesium ion by a carbonyl from the conserved Proline-Proline (“PP”) motif in Switch 1. Using our crystal structures, SAXS and molecular modeling, we propose a three-dimensional structural model of the monomeric and dimeric soluble domains of Miro1 at low resolution. The homo-dimeric model suggests that assembly is mediated via a conserved Serine-Glutamate-Leucine-Phenylalanine-Tyrosine-Tyrosine “SELFYY” motif in the nGTPase. We also characterize the putative Miro GTPase Activating Protein (GAP) called Vibrio outer protein E (VopE) using bioinformatic analysis, homology modeling, SEC-MALS, SEC-SAXS, and NMR spectroscopy. These data suggest that the VopE GAP domain forms an elongated 4-helix bundle similar to other bacterial GAP proteins, but with increased internal flexibility and disordered termini. Altogether, we show that Miro is structurally and functionally distinct from “on/off switch”-like GTPases and its putative GAP VopE is most homologous to the Yersinia outer protein E (YopE) in structure.

Creator