Dataset Information

Hydrogen Deuterium Exchange of MAF1 and RalGAP

ABSTRACT: A Toxoplasma gondii MAF1b - human RalGAPα1 complex highlights a potentially previously uncharacterized immunomodulatory role for MAF1b

INSTRUMENT(S): impact HD

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John Burke

LAB HEAD: John Burke

PROVIDER: PXD034607 | Pride | 2024-05-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	03_11_2017Cam_HDX2_RALGAP_MA1_DBSEARCH_3.rar	Other
	03_11_2017_Cam_HDX2_MAF1_DBSEARCH_3.rar	Other
	10_11_17_CamHDX2_M_MSMS_2_0_01_4915.d.rar	Other
	10_11_17_CamHDX2_R_MSMS_2_0_01_4912.d.rar	Other
	23_11_2017_CP_HDX2_M_0.3_1_0_01_4939.d.rar	Other

Items per page:

1 - 5 of 53

Publications

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

Powell Cameron J CJ Jenkins Meredith L ML Hill Tara B TB Blank Matthew L ML Cabo Leah F LF Thompson Lexie R LR Burke John E JE Boyle Jon P JP Boulanger Martin J MJ

The Journal of biological chemistry 20231221 1

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 ...[more]

PMID: 38141762

Similar Datasets

Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.