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. 2010 Aug 11;18(8):985-95.
doi: 10.1016/j.str.2010.05.013.

The RalB-RLIP76 complex reveals a novel mode of ral-effector interaction

R Brynmor Fenwick  1 , Louise J Campbell, Karthik Rajasekar, Sunil Prasannan, Daniel Nietlispach, Jacques Camonis, Darerca Owen, Helen R Mott
Affiliations

The RalB-RLIP76 complex reveals a novel mode of ral-effector interaction

R Brynmor Fenwick et al. Structure. .

Abstract

RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain, and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral-binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB, and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84, and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.

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Figures

Figure 1
Figure 1. Structures of RLIP76 GBD alone and in complex with RalB.
A. Domain structure of RLIP76. The approximate positions of the RhoGAP domain, the Ral binding domain (GBD) and coiled-coil region are represented as coloured boxes, the regions found to interact with AP2 and POB1 are indicated and the locations of the putative ATP binding sites are marked with red arrows (Awasthi et al., 2001). B. Structure of free RLIP76 GBD. On the left is the backbone trace of the family of structures consistent with the NMR restraints, on the right is the closest structure to the mean. All structure figures were produced using Molscript (Kraulis, 1991) and rendered with Raster3D (Merritt and Bacon, 1997). C. Structure of the RLIP76 GBD-RalB complex. Ral is shown in blue and RLIP76 is lilac. On the left is the backbone trace of the lowest energy structures, on the right is the closest structure to the mean.
Figure 2
Figure 2. Details of the interactions between RalB and the RLIP76 GBD
A. Interactions involving the RalB switch 1 and interswitch regions. RalB is shown in blue and the RLIP76 GBD is in lilac. The sidechains are shown in a ball-and-stick representation with sticks in the same colours as the ribbon for each molecule and the atoms coloured as follows: carbon, dark grey; oxygen, red; nitrogen blue. B. Interactions involving the RalB switch 2. The colour scheme is the same as in part (a). C. Summary of all interactions between the proteins. Putative hydrogen bonds and salt bridges are shown as dotted lines between the participating atoms. RalB is shown in blue and RLIP76 is shown in lilac. D. Interactions involving His-413RLIP76. Sidechains are shown in a spacefilling representation superimposed with a ball-and-stick. The colour scheme is the same as in part (a). E. Trp-430 of RLIP76 is surrounded by a cage of RalB sidechains. The colour scheme is the same as in part (a).
Figure 3
Figure 3. Comparison of free RalB·GMPPNP (pdb code 2KE5) and RLIP76 GBD with their structures in the complex. The closest structure to the mean is shown in each case.
A. The RalB-RLIP76 complex with the contributing free proteins overlaid. RalB is shown in blue (complex) and green (free), RLIP76 GBD is shown in lilac (complex) and yellow (free). The regions of greatest divergence in RalB, the switch regions and the interswitch hairpin, are labelled. B. The RLIP76 GBD alone, in the structures adopted in the free and complex forms, in an orientation that shows the small changes in the lengths and orientations of the α-helices. The structure of free RLIP76 GBD is yellow and that in the complex is lilac.
Figure 4
Figure 4. 31P NMR spectra recorded on RalB·GTP.
A. RalB·GTP recorded at 25°C. The phosphorus resonances for the GTP attached to RalB are labelled and were assigned as described previously (Fenwick et al., 2009). The resonances labeled ‘d’ are likely to be due to small amounts of contaminating GDP in the sample {Geyer, 1996 #1364}. B. The same sample recorded at −6°C. Each of the three phosphorus resonances is split into two at low temperatures and their positions are marked by dotted lines. Note that the GDP peak that is close to the α resonance contributes to the state 1 component of the α resonance at −9.94 ppm at low temperatures. This has the effect of making the state 1 component larger than the state 2 component (−10.93 ppm), whereas previously the state 2 component was larger {Fenwick, 2009 #1345}. C. The spectrum of RalB·GTP recorded in the presence of excess RLIP76 GBD at 25°C. D. The same sample recorded at −6°C. The splitting is still visible for the α and β resonances. Although the GDP peak is again contributing to the state 1 component of the α resonance, it cannot account for all of the intensity of the state 1 peak, since the GDP peak at −9.5 ppm is of a lower intensity than the peak at −1.0 ppm in this sample.
Figure 5
Figure 5. Sequence alignments.
A. Alignment of RLIP76 GBD from different organisms. The positions of α-helices in the structure are shown as grey cylinders on the top of the alignment. Residues that are conserved in all sequences are coloured white on a black background. Residues whose properties are conserved are boxed. The stars above the sequences mark the position of residues that interact with RalB. Hs – Homo sapiens; Mm – Mus musculus; Gg – Gallus gallus; Tn –Tetraodon nigroviridis; Xl – Xenopus laevis; Dr – Danio rerio; Is – Ixodes scapularis; Bf – Branchiostoma floridae; Dm – Drosophila melanogaster; Aa – Aedes aegypti; Am – Apis mellifera; Ce – Caenorhabditis elegans. B. Alignment of the N-terminal regions of RalA, RalB and Ha-Ras. The positions of α-helices in the Ral B structure are shown as grey cylinders and the β-strands as grey arrows, on the top of the alignment. Residues that are conserved between all three sequences are boxed. The stars above the sequences indicated the residues that interact with RLIP76 GBD.
Figure 6
Figure 6. Measurement of the affinities of Q72L RalB and selected mutants for the RLIP76 GBD.
The indicated concentration of [3H] GTP-labelled RalB were incubated with 80nM RLIP76 GBD-His in SPAs. The SPA signal was corrected by subtraction of a blank from which RLIP76 GBD-His was omitted. The effect of RalB on this corrected SPA counts/min signal was fitted to a binding isotherm to give an apparent Kd value and the signal at saturating concentrations of RalB. The data are expressed as a percentage of this maximum signal. The calculated Kds were: RalB Q72L, 183.9 ± 19.9nM; RalB Q72L K47A, 339.5 ± 34.7nM; RalB Q72L A48G, 945.5 ± 60.7; RalB Q72L L67A, >1μM; RalB Q72L A77R, 585.0 ± 50.2 nM; RalB Q72L I78A, 724.2 ± 55.1nM; RalB Q72L Y82A, >1μM.
Figure 7
Figure 7. Comparison of structures of Ral-effector complexes shows that they bind to overlapping interfaces of the G protein.
A. RalB-RLIP76: RLIP76 uses a coiled-coil and binds to both switch 1 and switch 2. RalB is blue and RLIP76 is lilac B. RalA-Exo84: Exo84 uses a PH domain and binds to both switch 1 and switch 2. RalA is purple and Exo84 is orange. C. RalA-Sec5: Sec5 uses an immunoglobulin-like domain and binds exclusively to switch 1 and the interswitch region. RalA is blue and Sec5 is red.
Figure 8
Figure 8. Displacement of [3H]GTP·RalB from GST-Sec5 by RLIP76.
Increasing concentrations of RLIP76 GBD were titrated into fixed concentrations of [3H]GTP·RalB (20nM) and GST-Sec5 GBD (20nM) in competition SPAs. The fit of the inhibition of the [3H]GTP·RalB/GST-Sec5 GBD interaction is shown and yields a Kd of 199.4 ± 33.3 nM. This is the same as the Kd measured by direct binding (Figure 6).
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