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. 2006 Jun 6;45(22):6826-34.
doi: 10.1021/bi052655o.

Molecular dissection of Rab11 binding from coiled-coil formation in the Rab11-FIP2 C-terminal domain

Jie Wei  1 , Sebastian Fain, Celia Harrison, Larry A Feig, James D Baleja
Affiliations

Molecular dissection of Rab11 binding from coiled-coil formation in the Rab11-FIP2 C-terminal domain

Jie Wei et al. Biochemistry. .

Abstract

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.

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Figures

Figure 1
Figure 1
Peptide sequences representing regions of FIP2 containing the coiled-coil domain. The parent construct, RH70, comprises amino acid residues 443 to 512 of FIP2. The italic letters above the sequences denote the predicted heptad repeats within the coiled-coil. Residues in the a and d positions are in bold and predicted to participate in the formation of a hydrophobic interface. The residues comprising the minimal Rab-11 binding domain (7) are underlined.
Figure 2
Figure 2
Helical wheel representation of residues of the RHCC dimer (453-489) in heptad repeats of a coiled-coil model.
Figure 3
Figure 3
Chemical cross-linking reveals that the coiled-coil forms a dimer. Purified RHCC domain (40 μM) was incubated with BS3 cross-linker for 30 min at 25 °C and the reaction was stopped by the addition of 0.1M Tris. Cross-linked products were analyzed by a 15% Tris-tricine gel. The arrow indicates the major cross-link product at MW8000.
Figure 4
Figure 4
Sedimentation equilibrium solute distribution for RHCC. Centrifugation was performed at various loading concentrations and two speeds. Data are shown for a concentration of 0.7 mg/ml collected at 35,000 rpm. The data were initially fitted with an equation for a single, ideal species but a better fit was obtained with a dimer-hexamer form of the equation. The line is the best fit through the data points (circles). The residuals are shown in the panel above the data. This fit was typical of data obtained with all sample concentrations of 1.4 mg/ml, 0.7 mg/ml, 0.35 mg/ml, and 0.175 mg/ml respectively at both centrifugation speeds (35,000 and 50,000 rpm). The global fit from all eight data sets was consistent with a dimer-hexamer two state model with a predominant dimer (>95%) with a molecular weight of 9957 Da.
Figure 5
Figure 5
The thermal stability increases by introducing a disulfide-bridge into the C-terminus of the FIP2 coiled-coil domain. Data shows the temperature denaturation profile of RHCC (squares) and RHcys (circles). Molar ellipticity data were fit to obtain Tm values. The Tm was 48 °C for RHCC (at a peptide concentration of 0.11 mg/ml) and 71 °C for RHcys. Samples were prepared in 10 mM Tris buffer, pH 7.3.
Figure 6
Figure 6
Isothermal titration calorimetry of various forms of the FIP2 coiled-coil domain with Rab11. Each experiment was conducted with 100 μM FIP2 domains injected into 10 μM Rab11. The raw data is shown in red and the integrated area of each peak and the non-linear, least square fit curve representing the heat released is shown in black. (A) RH50 injected into Rab11-GMP-PNP. Ka = (1.18 ± 0.25) × 106 M-1, ΔH = -39.5 ± 1.0 kcal/mol, TΔS = -31.1 kcal/mol, n=0.37. (B) RHCC injected into Rab11-GMppNp. (C) RHNQ injected into Rab11-GMP-PNP. Ka = (1.21± 0.25) × 106 M-1 ΔH = -25.8 ± 1.1 kcal/mol, TΔS = -18.4 kcal/mol, n=0.36.
Figure 7
Figure 7
Comparison of different constructs by circular dichroism. Data shows circular dichroism spectra of RH50 (triangles), RHCC (squares), and RHNQ (circles). Spectra were recorded at 25°C with a peptide monomer concentration of 11 μM in 10 mM Tris buffer, pH 7.3.
Figure 8
Figure 8
Circular dichroism spectra show induced helical content in the complexes formed by FIP2 coiled-coil domains and Rab11. (A: RH50, B: RHNQ). Squares represent the peptides, circles represent the 1:1 molar mixture of Rab11 and peptide, triangles represent Rab11, and plus signs represent the sum of the CD signals from peptide and Rab11. All spectra were recorded at 25°C in 10 mM Tris buffer, pH 7.3.
Figure 9
Figure 9
A model of FIP2 binding to Rab11. The models show the interactions between various FIP2 coiled-coil domains and Rab11. A: RH50. The pre-formed dimer of RH50 binds Rab11; B: RHNQ. RHNQ does not have a pre-formed coiled-coil, but binds Rab11 as well as RH50 with significant conformational changes; C: RHCC. RHCC is a well-structured coiled-coil that does not have appreciable affinity for Rab11.
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