Biophysical analysis of the interaction of Rab6a GTPase with its effector domains.
Rab GTPases are key regulators of intracellular vesicular transport that control vesicle budding, cargo sorting, transport, tethering, and fusion. In the inactive (GDP-bound) conformation, Rab GTPases are targeted to intracellular compartments where they are converted into the active GTP-bound form and recruit effector domain containing proteins. Rab6a has been implicated in dynein-mediated vesicle movement at the Golgi apparatus and shown to interact with a plethora of effector proteins. In this study, we identify minimal Rab6a binding domains of three Rab6a effector proteins: PIST, BicaudalD2, and p150(glued). All three domains are >15-kDa fragments predicted to form coiled-coil structures that display no sequence homology to each other. Complex formation with BicaudalD2 and p150 has a moderate inhibitory effect on the intrinsic GTPase activity of Rab6a, while interaction with PIST has no influence on the hydrolysis rate. The effectors bind activated Rab6a with comparable affinities with K(d) values ranging from high nanomolar to low micromolar. Transient kinetic analysis revealed that effectors bind to Rab6a in an apparent single-step reaction characterized by relatively rapid on- and off-rates. We propose that the high off-rates of Rab6.effector complexes enable GTPase-activating protein-mediated net dissociation, which would not be possible if the off-rate were significantly slower.
Bergbrede
Tim
T
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Chuky
Nam
N
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Schoebel
Stefan
S
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Blankenfeldt
Wulf
W
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Geyer
Matthias
M
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Fuchs
Evelyn
E
Cancer Research Centre, University of Liverpool, Liverpool L3 9TA, United Kingdom.
Goody
Roger S
RS
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Barr
Francis
F
Cancer Research Centre, University of Liverpool, Liverpool L3 9TA, United Kingdom.
Alexandrov
Kirill
K
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: k.alexandrov@imb.uq.edu.au.
D001703:Biophysics; D002151:Calorimetry; D002850:Chromatography, Gel; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
C094135:Rab6 protein; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins