Fig 1: Molecular basis of Arl5B(GTP) binding to ARMH3.A, zoom-in on the predicted ARMH3-Arl5B(GTP) interface, showing select contact residues of ARMH3 and Arl5B labeled and shown as sticks. Switch regions of Arl5B are coloured pink. B, multiple sequence alignments of ARMH3 and Arl5 from H. sapiens (human), M. musculus (mouse), D. rerio (zebrafish), and D. melanogaster (fruit fly). Secondary structures of ARMH3 and Arl5 are annotated above the alignment, while the HD exchange differences described in Fig. 1 are annotated below the ARMH3 alignment. Predicted contact residues with >5 Å2 of buried surface area are annotated using arrows. C, zoomed in view of the ARMH3-Arl5B(GTP) interface with key residues and mutations labelled. D, biolayer interferometry (BLI) association and dissociation curves of ARMH3 and Arl5B(GTPγS) mutants compared to wild-type. His-Arl5B(GTPγS) was loaded onto the anti-penta His tip at 25 nM and dipped into ARMH3 at 100 nM.
Fig 2: Biolayer interferometry and HDX-MS reveal canonical GTPase-effector interaction.A, domain schematics of full-length ARMH3 and Arl5. Constructs used in this paper are Arl5A(15–179) and Arl5B(15–179), which are referred to as Arl5A and Arl5B throughout this paper. B, schematic of the biolayer interferometry (BLI) assay showing binding of immobilised His-Arl5 on the tip to full-length ARMH3 in solution. C, BLI traces of Arl5B(GTPγS) and Arl5B(GDP) binding to full-length ARMH3. His-Arl5B(GTPγS) or His-Arl5B(GDP) was loaded onto the anti-penta His tip at 25 nM and dipped into ARMH3 at 100 nM. D, comparison of BLI traces generated from Arl5B(GTPγS) and Arl5A(GTPγS) interacting with ARMH3. His-Arl5B(GTPγS) or His-Arl5A(GTPγS) was loaded onto the anti-penta His tip at 25 nM and dipped into ARMH3 at 100 nM. E, dose response of Arl5B(GTPγS) binding to ARMH3. His-Arl5B(GTPγS) was loaded onto the anti-penta His tip at 25 nM and dipped into ARMH3 (15–100 nM). All curves were fit with a partial, 1:1 binding model. The KD value reported was generated using the average KD given for each curve meeting inclusion criteria, error is reported as standard deviation (n = 5). F, sum of the number of deuteron differences in ARMH3 upon binding to Arl5B(GTPγS) analyzed over the entire deuterium exchange time course. Each point represents the centre residue of an individual peptide. Peptides that met the significance criteria (defined as >0.4 Da, >5%, and p < 0.01 in an unpaired two-tailed t test at any time point) are coloured red. Error is shown of the sum of standard deviations (SDs) across all five time points (n = 3). G, selected deuterium exchange time courses that showed significant decreases in exchange upon complex formation. Error is shown as SD (n = 3). H, AlphaFold3 prediction of ARMH3 in complex with Arl5B and co-factors GTP and Mg2+ coloured by chain, important structural features of ARMH3 and Arl5B are annotated. I, predicted aligned error (pae) plot of the AlphaFold3 prediction of the ARMH3-Arl5B(GTP) complex with co-factors GTP and Mg2+. J, AlphaFold3 model of the ARMH3-Arl5B complex coloured by significant decreases in deuterium exchange in ARMH3 upon binding to Arl5B(GTPγS). Boxes highlight significant peptides at the ARMH3-Arl5B interface and distal regions of ARMH3 showing allosteric changes.
Supplier Page from DNASU for ARL5A (Homo sapiens) in pDNR-Dual (Creator donor/master vector)