RLIP76: A Structural and Functional Triumvirate

Abstract

RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may mediate membrane association, consistent with its role in transport. RLIP76 is an ATP-dependent transporter with ATP-binding sites within the N-terminus and the Ral binding domain. Furthermore, RLIP76 is subject to extensive phosphorylation, particularly in the N-terminal region. In contrast, the C-terminal region is thought to form an extensive coiled-coil that could mediate dimerization. Here, we review the structural features of RLIP76, including experimental data and computational predictions, and discuss the implications of its various post-translational modifications.

Keywords: RLIP76; Ral; RalBP1; RhoGAP; small G protein.

Figure 1

Architecture and conservation of the domains in RLIP76. (A) Domain architecture of RLIP76. The central RhoGAP and Ral binding domains are flanked by structurally uninvestigated N- and C-termini. The C-terminus is predicted to form a coiled coil, while the N- terminus is predicted to be disordered. The positions of two ATP binding sites are indicated by inverted triangles. Known interactions are labelled, with their approximate binding regions indicated by arrows. (B) Phylogenetic tree of RLIP76 in a subset of organisms. The schematic (styled as in Figure 1A) shows the conserved features for each organism. Only the RhoGAP and RBD are conserved in Caenorhabditis elegans. The predicted coiled-coil is present in Danio rerio through to Homo sapiens, while the two ATP binding motifs are present in Rattus norvegicus, Mus musculus and Homo sapiens.

Figure 2

Structures of the free RBD and the RBD-RalB complex. (A) Comparison of the free RBD (cyan) superimposed on the RBD RalB complex (RBD = orange, RalB = green) (2KWH, 2KWI). The helix in the bound RBD extends, with Gln417 pointing in the same direction as His413. Interactions involving His413 and Gln417 are shown with the sidechains in spacefilling representation superimposed with sticks: carbons are the same colour as the ribbon, oxygen is red and nitrogen is blue; (B) Comparison of the hydrophobic pocket of WT RBD and the tighter binding E427H/Q433L/K440R mutant (6ZQT). Residues in the hydrophobic pocket round Trp430 is shown with the sidechains in spacefilling representation superimposed with sticks. Colour scheme is the same as in (A).

Figure 3

RLIP76 RhoGAP and RBD interactions. RLIP76 could interact simultaneously with both Ral and Rho family proteins. Cdc42 was docked to the RLIP76 didomain (2MBG) using the Cdc42-p50RhoGAP complex to overlay the RhoGAP domains (1AM4). The RBD of RLIP76 of this model was then superimposed with the RBD in the RalB complex (2KWI). The RLIP76 RBD is orange, the RhoGAP domain is cyan, the RLIP76 linker is blue, RalB is green and Cdc42 is yellow. Helices in RalB and Cdc42 that would contact the membrane in this orientation are labelled and their C-terminal lipid modifications are shown in red.

Figure 4

Predicted disorder of RLIP76. (A) Hydropathy plot of RLIP76 N- and C-termini (green diamond and red triangle, respectively), the remaining plotted proteins (ordered, blue and disordered, orange) are from the PONDR data set. RLIP76 lies to the left of the boundary line for disordered proteins. (B) PONDR plot of disorder across residues 1–655 (full length) RLIP76 (top). IUPRED plot of disorder (blue) and ANCHOR score (orange) across residues 1–655 (bottom). In both PONDR and IUPRED scores above 0.5 are disordered. ANCHOR scores above 0.5 predict regions that are likely to fold upon binding. Grey boxes highlight the locations of the RhoGAP and RBD.

Figure 5

COILS prediction of RLIP76. Plot of predicted coil coiled regions in RLIP76 by COILS server. Window size is the number of amino acids in the sliding window the server uses across the sequence. The resolution between globular and coiled-coil score distributions decreases strongly with decreasing window size. However, ends of coiled coils and short sections can be identified with smaller windows.

Figure 6

Compiled knowledge of the structure of RLIP76. Phosphorylation sites are shown as maroon lollipops. Predicted and known regions of structure are boxed. The predicted palmitoylation site is shown as a red zig-zag. The poly-lysine tract and predicted molecular recognition features are also shown.