Domain structures of INPP5B and OCRL and their F&H motif-containing interactors, and the crystal structure of the human ASH-RhoGAP domain in complex with the F&H peptide from human Ses1. (a) The ASH domain is colored in red, the RhoGAP domain in blue, and the Ses1 F&H peptide in yellow. Molecular graphics for this and all subsequent figures were generated with Pymol⁴². (b) Close-up view of the interface between the F&H peptide (yellow) and the RhoGAP domain (blue). (c) F&H peptide sequences used in this study. Phosphoserine residues are indicated in green. (d) GST-ASH-RhoGAP constructs of OCRL were assayed for binding to APPL1 from rat brain homogenate. The GST negative control is marked as a -, and the OCRL constructs are either wild-type (WT), or mutated in the F&H binding site (W739A and D743R). As predicted, the mutants disrupt binding to APPL1, without affecting binding to clathrin heavy chain (CHC).

Mutation of the F&H binding site in OCRL disrupts its colocalization with F&H motif-containing proteins. Colocalization of exogenously expressed OCRL and F&H motif-containing proteins was assessed in human fibroblasts derived from a patient with Lowe Syndrome ³⁷. Images are shown in the top panels, with quantitation below. (a) Mutation of the F&H interface (W739A) disrupts colocalization of RFP-APPL1 and GFP-OCRL on individual endosomes, although the overall localization of the two proteins is not compromised. Quantification of the colocalization of APPL1 with OCRL^WT and OCRL^W739A is expressed as the percentage of APPL1 colocalizing with the OCRL protein. (b) mTagRFP-T-Ses2 expressed in the absence of co-transfected OCRL is primarily cytosolic. When co-transfected with GFP-OCRL^WT, mTagRFP-T-Ses2 colocalizes with this protein on endosomes and in the Golgi complex area, but this colocalization is disrupted by the W739A mutation in OCRL. The quantification of colocalization is expressed as a function of the colocalization in well-defined puncta, as the main result of the mutation is a diffuse signal of the Ses2 protein, and errors are the SEM.

Conserved surfaces of the ASH-RhoGAP domain map to the F&H interaction and potential Rab interaction surfaces. (a) Surface representation is colored by degree of conservation with the darker colors indicating the most conservation. Images were generated using PROTSKIN⁴³. (b) Sequence alignment highlighting the conservation of the major residues responsible for recognition of the F&H motif. Accession codes for sequences used in the alignment are listed in Supplementary information.

Patient mutations affecting F&H binding are global folding mutations. Patient mutations are represented in all structures as spheres. (a) The mapping of the patient missense mutations onto the ASH-RhoGAP structure, with two mutation networks expanded, one in the ASH domain and the other in the RhoGAP domain. (b) F&H binding is disrupted due to destabilization of OCRL. Inspection of a coomassie-stained gel shows significant degradation as well as the co-purification of a DnaK chaperone for mutants that display a lack of F&H binding. This destabilization is not seen in our designed F&H-binding mutation (W739A), a Rab5-defective mutant (F668V), or a splice-site mutation that results in a lack of expressed protein (A861T). Patient mutations which give rise to Dent 2 disease are indicated by an asterisk. (c) The GST-ASH-RhoGAP OCRL constructs in panel b were assessed for APPL1 binding using a GST pulldown assay from rat brain followed by western blot for APPL1.

Characterization of a Rab5 binding-deficient mutation. (a) The residues shown to be important for Rab binding both in previous studies¹⁹, as well as in this one (Phe668), are represented as gray spheres. (b) Pull-downs using nucleotide-loaded Rab5 as bait for over-expressed OCRL constructs show that the GTPγS-dependent interaction between Rab5 and OCRL is perturbed by the F668V mutation, while not affected by mutation of the F&H recognition site.