Biochemical and Structural Studies of the Outer Mitochondrial Membrane Protein, Miro1
Creators: name: Eberhardt, Emily
Publisher: name: My University
Year: 2023
Subjects: subject: miro
subject: mitochondrial trafficking
subject: mitochondrial dynamics
subject: miro oligomerization
subject: miro structure
subject: Molecular, Cellular and Developmental Biology
subject: Science

description: Mitochondrial trafficking is an essential process that must be regulated to meet the vast energy needs of the cell. This process is especially important in neurons, as mitochondria must be transported across an extensive distance through heavily polarized and branched dendritic processes. Failure to regulate mitochondrial trafficking is detrimental and results in various neurological conditions, such as Parkinson’s disease. The mitochondrial trafficking complex consists of the microtubule motor proteins kinesin and cytoplasmic dynein, which interact with the cargo adaptor protein, trafficking kinesin protein (TRAK), to link the microtubule tracks to mitochondria. Tethered to the outer mitochondrial membrane, the small G protein Miro coordinates association with the motor-adaptor complex. Miro is a unique protein containing two distinct GTPase domains flanking two calcium-binding EF-Hands, tethered to the mitochondria through a small transmembrane tail. The N-terminal GTPase (NGTPase) of Miro is critical for regulating mitochondrial trafficking. NGTPase mutations decrease or halt mitochondrial trafficking, although the mechanism for how this occurs remains unclear. Finally, while these characteristic mutations (constitutively active/GTP-bound: Miro1P13V and dominant negative/GDP-bound: Miro1T18N) have been used to study the GTPase domains of Miro in cells, there are limited biochemical studies of the effects of these mutations on the activity, function, or structure of Miro. In this study, I utilized in vitro reconstitution to study the molecular mechanism of Miro1 domain regulation. First, I determined that locking the Miro1 NGTPase in the GTP-bound state (Miro1P13V) results in the formation of discrete higher-ordered species (dimers, trimers, and tetramers) that have a decreased affinity for TRAK1. Negative stain electron microscopy showed that the higher-ordered species formed through association at multiple interaction points. Additionally, I demonstrated that TRAK1 has two Miro1-binding sites, implying multiple regions on TRAK1 coordinate the regulation of mitochondrial trafficking. Throughout my studies, I made significant advances in the biochemical study of the mitochondrial trafficking protein complex. First, I solubilized full-length Miro1 and made steps toward full-length TRAK1 solubilization, which will be valuable assets for future biochemical studies. Next, I optimized a reliable thermal shift assay to indicate that GTP stabilizes Miro1. This assay will be utilized to further study the effects of ligands (such as GDP, GMP-PNP, and Ca2+) on Miro1 domains. Finally, I used cryogenic electron microscopy to study the structure of Miro1, generating three-dimensional (3D) reconstructions of soluble Miro1 at sub-nanometer resolution (8.4 Å). Together, these studies provide an essential framework for future research into many aspects of Miro1 regulation. These aspects include the impact of nucleotide (GTP) binding on domain activity and structure, GTPase activity, interaction with TRAK1, and the role of full-length Miro1 in the outer mitochondrial membrane. Further studies directed at understanding Miro1- mediated mitochondrial trafficking will allow for determining therapeutic targets for disease intervention.