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Recognition of the F&H motif by the Lowe syndrome protein OCRL

Nature Structural & Molecular Biology volume 18, pages 789–795 (2011)Cite this article

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Abstract

Lowe syndrome and type 2 Dent disease are caused by defects in the inositol 5-phosphatase OCRL. Most missense mutations in the OCRL ASH-RhoGAP domain that are found in affected individuals abolish interactions with the endocytic adaptors APPL1 and Ses (both Ses1 and Ses2), which bind OCRL through a short phenylalanine and histidine (F&H) motif. Using X-ray crystallography, we have identified the F&H motif binding site on the RhoGAP domain of OCRL. Missense mutations associated with disease affected F&H binding indirectly by destabilizing the RhoGAP fold. By contrast, a disease-associated mutation that does not perturb F&H binding and ASH-RhoGAP stability disrupted the interaction of OCRL with Rab5. The F&H binding site of OCRL is conserved even in species that do not have an identified homolog for APPL or Ses. Our study predicts the existence of other OCRL binding partners and shows that the perturbation of OCRL interactions has a crucial role in disease.

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Figure 1: 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.
Figure 2: Mutation of the F&H binding site in OCRL disrupts its colocalization with proteins that contain F&H motifs.
Figure 3: Conserved surfaces of the ASH-RhoGAP domain map to the F&H-interaction and potential Rab-interaction surfaces.
Figure 4: Disease-causing mutations that affect F&H binding are global folding mutations.
Figure 5: Characterization of a mutation that impairs binding of Rab5.

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Acknowledgements

We thank J. Murphy (Yale School of Medicine Macromolecular Crystallography Facility) and K. Reinisch for advice and assistance; the Yale School of Medicine W.M. Keck small-scale peptide synthesis facility for peptide synthesis; M. Stagi for computational support; and D. Balkin and H. Sondermann for discussion. This work has been supported by the following funding sources: Browne-Cox postdoctoral fellowship (M.P.), US National Institutes of Health DK45735 and DK082700 (P.D.C.), DA018343 (E.F.-S. and P.D.C.), and grants from the Lowe Syndrome Association (P.D.C.) and the Lowe Syndrome Trust (P.D.C. and L.S.). The Biacore T100 instrumentation, and the size exclusion chromatography and light scattering instrumentation (SEC/LS system), were supported by US National Institutes of Health grants 1S10RR026992 and 1S10RR023748, respectively.

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  1. Michelle Pirruccello and Laura E Swan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA

    Michelle Pirruccello, Laura E Swan & Pietro De Camilli

  2. Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA

    Michelle Pirruccello, Laura E Swan & Pietro De Camilli

  3. Program in Cellular Neuroscience Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut, USA

    Michelle Pirruccello, Laura E Swan & Pietro De Camilli

  4. W.M. Keck Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, Connecticut, USA

    Ewa Folta-Stogniew

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Contributions

M.P., L.E.S. and P.D.C. designed research; M.P., L.E.S. and E.F.-S. conducted experiments and contributed new reagents or analytical tools; M.P., L.E.S. and P.D.C. analyzed data; M.P. and P.D.C. wrote the paper.

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Correspondence to Pietro De Camilli.

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Pirruccello, M., Swan, L., Folta-Stogniew, E. et al. Recognition of the F&H motif by the Lowe syndrome protein OCRL. Nat Struct Mol Biol 18, 789–795 (2011). https://doi.org/10.1038/nsmb.2071

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