Molecular dissection of Rab11 binding from coiled-coil formation in the Rab11-FIP2 C-terminal domain.
The Rab11-family interacting protein (Rab11-FIP) group of effector proteins contain a highly conserved region in their C-termini that bind the GTPase, Rab11. Rab11 belongs to the largest family of small GTPases and is believed to regulate vesicle docking with target membranes and vesicle fusion. The amino acid sequence of the Rab11-FIP proteins predicts coiled-coil formation in the conserved C-terminal domain. In this study on Rab11-FIP2, we found experimental evidence for the coiled-coil and then defined the minimal structured core using limited proteolysis. We also showed that the Rab11-FIP2 coiled-coil domain forms a parallel homodimer in solution using cross-linking and mutagenesis and sedimentation equilibrium experiments. Various constructs representing the C-terminal domain of Rab11-FIP2 were characterized by circular dichroism, and their affinity with Rab11 was measured using isothermal titration calorimetry. The longest construct was both well-structured and bound Rab11. A construct truncated at the N-terminus was poorly structured but retained the same affinity for binding to Rab11. Conformational changes were also demonstrated upon complex formation between Rab11 and Rab11-FIP2. A construct truncated at the C-terminus, which was the minimal coiled-coil domain defined by limited proteolysis, did not retain the ability to interact with Rab11, although it was as well-structured as the longer peptide. These data show that coiled-coil formation and Rab11 binding are separable functions of the C-terminal domain of Rab11-FIP2. The dissection of Rab11 binding from the formation of defined structure in a coiled-coil provides a potential mechanism for regulating Rab11-dependent endosomal trafficking.
Wei
Jie
J
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Fain
Sebastian
S
Harrison
Celia
C
Feig
Larry A
LA
Baleja
James D
JD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D000099193:rab11 GTP-Binding Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D014357:Trypsin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C463963:RAB11FIP2 protein, human