Toxoplasma gondii mitochondrial association factor 1b interactome reveals novel binding partners including Ral GTPase accelerating protein α1

Cameron J Powell; Meredith L Jenkins; Tara B Hill; Matthew L Blank; Leah F Cabo; Lexie R Thompson; John E Burke; Jon P Boyle; Martin J Boulanger

doi:10.1016/j.jbc.2023.105582

Toxoplasma gondii mitochondrial association factor 1b interactome reveals novel binding partners including Ral GTPase accelerating protein α1

J Biol Chem. 2024 Jan;300(1):105582. doi: 10.1016/j.jbc.2023.105582. Epub 2023 Dec 21.

Authors

Cameron J Powell¹, Meredith L Jenkins¹, Tara B Hill¹, Matthew L Blank², Leah F Cabo², Lexie R Thompson¹, John E Burke³, Jon P Boyle², Martin J Boulanger⁴

Affiliations

¹ Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
² Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
³ Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada; Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
⁴ Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada. Electronic address: mboulang@uvic.ca.

Abstract

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a K_d of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness.

Keywords: GTPase activating protein (GAP); RalGAPα1; Toxoplasma gondii; host mitochondrial association; host-pathogen interaction; hydrogen exchange mass spectrometry; hydrogen-deuterium exchange; isothermal titration calorimetry (ITC); molecular modelling.

MeSH terms

Binding Sites
Calorimetry
Chromatography, Gel
Fibroblasts / metabolism
Fibroblasts / parasitology
GTPase-Activating Proteins* / chemistry
GTPase-Activating Proteins* / genetics
GTPase-Activating Proteins* / metabolism
Humans
Hydrogen Deuterium Exchange-Mass Spectrometry
Mitochondria* / metabolism
Mitochondria* / parasitology
Protein Interaction Maps*
Protozoan Proteins* / chemistry
Protozoan Proteins* / genetics
Protozoan Proteins* / metabolism
Toxoplasma* / chemistry
Toxoplasma* / genetics
Toxoplasma* / metabolism
Two-Hybrid System Techniques

Substances

GTPase-Activating Proteins
Protozoan Proteins
RALGAPA1 protein, human