Dataset Information

Detailing the Structural and Dynamic Interactions Between WDR44 and Rab11

ABSTRACT: Analysis of the interactions between WDR44 and Rab11 utilizing HDX-MS

INSTRUMENT(S): impact HD

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John Burke

LAB HEAD: Dr. John E. Burke

PROVIDER: PXD032837 | Pride | 2023-02-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	02_07_22_NHHDX7_PA_0.3s_1_0_01_18776.d.zip	Other
	02_07_22_NHHDX7_PA_ND_0_01_18770.d.zip	Other
	02_07_22_NHHDX7_PC_0.3s_1_0_01_18780.d.zip	Other
	02_07_22_NHHDX7_R_ND_0_01_18774.d.zip	Other
	02_07_22_NHHDX7_UA_0.3s_1_0_01_18778.d.zip	Other

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Publications

Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11.

Thibodeau Matthew C MC Harris Noah J NJ Jenkins Meredith L ML Parson Matthew A H MAH Evans John T JT Scott Mackenzie K MK Shaw Alexandria L AL Pokorný Daniel D Leonard Thomas A TA Burke John E JE

The Journal of biological chemistry 20221201 1

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling ...[more]

PMID: 36463963

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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.