Membrane Curvature Protein Exhibits Interdomain Flexibility and Binds a Small GTPase

Gordon J King,Shu-Hong Hu,Jagath R Junutula,Wilko G.A Duprez,Jennifer L Martin,Russell J Jarrott,David E James,Jacqueline Stöckli,Christopher C Meoli,Brit Winnen,Andrew E Whitten

doi:10.1074/jbc.m112.349803

Abstract

The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a K(d) of 140 nM. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.

Highlights

APPL2 is an endosomal Rab effector forming part of a signaling pathway linking cell surface and nucleus
The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain
Helix 4 is separated from helix 3 by two residues forming a kink that orients the long axes of the two helices ϳ120o relative to each other

Summary

Background

APPL2 is an endosomal Rab effector forming part of a signaling pathway linking cell surface and nucleus. The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. We describe the first structural information on APPL2, the BARPH domains of human APPL2 (hAPPL2BARPH) We report both the crystal structure and SAXS solution structure of APPL2, showing unexpectedly that there is flexibility in the arrangement of the BAR and PH domains. We identify by yeast two-hybrid screen that Rab is a binding partner for APPL2, and we establish the thermodynamics and stoichiometry of the interaction between hAPPL2BARPH and Rab

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION