Membrane curvature protein exhibits interdomain flexibility and binds a small GTPase.
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.
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
Gordon J
GJ
King
Jacqueline
J
Stöckli
Shu-Hong
SH
Hu
Brit
B
Winnen
Wilko G A
WG
Duprez
Christopher C
CC
Meoli
Jagath R
JR
Junutula
Russell J
RJ
Jarrott
David E
DE
James
Andrew E
AE
Whitten
Jennifer L
JL
Martin
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D012997:Solvents / Q000737:chemistry; D055672:Static Electricity; D013499:Surface Properties; D014965:X-Rays; D020691:rab GTP-Binding Proteins / Q000378:metabolism
C464801:APPL1 protein, human; C528555:APPL2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D010716:Phosphatidylinositols; C484190:RAB31 protein, human; D012997:Solvents; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; D020691:rab GTP-Binding Proteins