ocrl rhogap domain  (Cytoskeleton Inc)

Journal: Nature structural & molecular biology

Article Title: Recognition of the F&H motif by the Lowe Syndrome protein OCRL

doi: 10.1038/nsmb.2071

Figure Lengend Snippet: 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 Pymol . (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).

Article Snippet: The ASH-RhoGAP domain of OCRL (residues 536–901 of human OCRL) was expressed in BL21 DE3 pRLIP (Promega) cells as a GST-fusion (pGex6P1, GE Healthcare Lifesciences) and purified with glutathione sepharose resin (GE Healthcare Lifesciences) in 20 mM Tris pH 8, 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM DTT.

Techniques: Generated, Construct, Binding Assay, Negative Control

Journal: Nature structural & molecular biology

Article Title: Recognition of the F&H motif by the Lowe Syndrome protein OCRL

doi: 10.1038/nsmb.2071

Figure Lengend Snippet: 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.

Article Snippet: The ASH-RhoGAP domain of OCRL (residues 536–901 of human OCRL) was expressed in BL21 DE3 pRLIP (Promega) cells as a GST-fusion (pGex6P1, GE Healthcare Lifesciences) and purified with glutathione sepharose resin (GE Healthcare Lifesciences) in 20 mM Tris pH 8, 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM DTT.

Techniques: Binding Assay, Mutagenesis, Staining, Copurification, Construct, GST Pulldown Assay, Western Blot