Fig 2: Validation of the WDR44–Rab11a interface.A, zoomed in view of the Rab11–WDR44 model with interfacial residues labeled. Rab11 is colored in smudge with switch I/II colored in orange and WDR44 in silver. B, alignment of WDR44 Rab-binding domain between eukaryotic model organisms (Homo sapiens, Mus musculus, Xenopus laevis, Danio rerio, and Drosophila melanogaster). Putative contact residues within 4 Å of Rab11 determined using the AlphaFold complex structure are colored blue. Evolutionarily conserved residues that were mutated and tested in C and D are highlighted with an ∗. C, normalized BLI responses of various WDR44 mutants binding Rab11 (25 nM WDR44, with 75 nM Rab11). Responses were normalized to the mean wildtype response. Two-tailed p values are represented as follows: ∗∗<0.001 and ∗<0.01. Error bars represent SD (n = 3). D, raw BLI association and dissociation curves of each mutant binding Rab11. About 25 nM WDR44 (334–389) mutants were immobilized onto fiber optic anti–penta-His tips and dipped into Rab11 at 75 nM. BLI, biolayer interferometry; WDR44, WD repeat–containing protein 44.

Fig 3: Phosphorylation of WDR44 stabilizes part of the Rab11-binding interface of WDR44.A, significant differences in HDX upon phosphorylation of apo-WDR44 are mapped onto the AlphaFold model of WDR44 alone according to the legend (top). The number of deuteron differences for all analyzed peptides over the entire exchange time course for WDR44 compared with phosphorylated WDR44. Each point represents the central residue of an individual peptide. Red indicates a peptide with a significant difference, and black indicates no significant difference. For all panels, error bars show SD (n = 3) (bottom). B, significant differences in HDX upon Rab11 incubation with phosphorylated WDR44 (334–504) are mapped onto the AlphaFold model according to the legend. C, deuterium incorporation curves for selected WDR44 and Rab11 peptides highlighting the differences upon complex formation with phosphorylated and unphosphorylated WDR44 (top). The number of deuteron differences for all analyzed peptides over the entire exchange time course for phosphorylated WDR44 compared with phosphorylated WDR44 in complex with Rab11. Each point represents the central residue of an individual peptide. Red indicates a peptide with a significant difference, and black indicates no significant difference. Error bars show SD (n = 3) (bottom). HDX, hydrogen/deuterium exchange; WDR44, WD repeat–containing protein 44.

Fig 4: Model of full-length WDR44 and defining the minimal RBD fragment and its affinity for Rab11.A, full-length WDR44 AlphaFold2 structure from the AlphaFold structure database (https://alphafold.ebi.ac.uk/) (23) with pLDDT <50 removed. Domains are colored according to B with the RBD colored in tan. Validation details are provided in Fig. S1C. B, the full-length domain schematic of WDR44 as well as fragments used to examine Rab11 binding. S344 is the site of phosphorylation. Domains are colored to match the AlphaFold structure in A. C, biolayer interferometry (BLI) association and dissociation traces of the shortest WDR44 (340–389) and longest WDR44 (334–504) fragments binding Rab11. Experiments were carried out using 25 nM or each WDR44 construct, with 150 nM of GTPγS-loaded Rab11. WDR44 protein was immobilized on the tip using a anti-His antibody (see schematic in D). D, cartoon schematic of BLI analysis of the binding of immobilized His-WDR44 (340–389) to Rab11a. E, association and dissociation curves for the dose response of His-WDR44 binding to varying Rab11 concentrations (10–1000 nM). Each experiment was carried out in triplicate, with all data graphed. F, normalized BLI response with varying concentrations of Rab11, with KD estimated (red line) by a one-site binding nonlinear regression. Each data point is shown (n = 3). RBD, Rab-binding domain; WDR44, WD repeat–containing protein 44.

Fig 5: WDR44 can be phosphorylated efficiently by Sgk3 specifically at S344.A, schematic of WDR44 (334–504) phosphorylation of S344 in vitro using Sgk3. B, peptide spectra of the unphosphorylated (left) and phosphorylated (right) WDR44 peptide, with S344 highlighted in red in the sequence. C, graph showing the intensity of unphosphorylated (top) and phosphorylated (bottom) peptide according to a dose response of Sgk3. D, MS intensity of phosphorylated (red) and unphosphorylated (black) peptide for wildtype and S344D WDR44 treated with 5 μg Sgk3. E, ratio of phosphorylated to unphosphorylated WDR44 graphed against Sgk3 kinase concentration from data in C. MS, mass spectrometry; WDR44, WD repeat–containing protein 44.