The disordered N-terminus of RLIP76: a hub for protein and membrane interactions

Change log

RLIP76 is an effector for the Ral family of small GTPases. Ral proteins are themselves downstream of Ras, and Ras mutations drive around 20% of all cancers. RLIP76 has been implicated in a number of cell processes ranging from receptor mediated endocytosis, to mitochondrial fission and metabolite transport. Despite implications in multiple signalling pathways, RLIP76 only has two defined domains, the Ral binding domain and a RhoGAP domain. The C-terminal third of RLIP76 is predicted to form a coiled-coil, whilst the N- terminus of RLIP76 was, in this work, observed by CD, SAXS and NMR to be intrinsically disordered. A multi-faceted approach, including traditional proton-detected triple-resonance NMR experiments and 13C direct-detection experiments, enabled the sequence-specific as- signment of 98% of the resonances in the disordered N-terminus, which then allowed for further structural and dynamic characterisation. Secondary chemical shifts, paramagnetic relaxation enhancement, and relaxation measurements describe a flexible protein with some secondary structural elements. Inspection of the protein sequence reveals lysine-rich regions within the N-terminal third of RLIP76, reminiscent of poly-basic membrane binding motifs. Attachment of RLIP76 directly to membranes, rather than localisation via other proteins, has never been assessed. Liposomes were therefore used as an in vitro tool to investigate mem- brane binding. The ability of RLIP76 to bind liposomes was quantified using sedimentation assays, turbidity assays and NMR. Binding was found to be dependent on the presence of anionic phospholipids and facilitated by the lysine-rich N-terminus. Protein-protein interac- tions with the N-terminus were then investigated, and the effect of membrane-attachment on the catalytic ability of the RhoGAP domain was explored.

Mott, Helen
Small GTPase, Disordered Proteins, Membrane, NMR
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Cambridge Trust / Wolfson College, Instruct-ERIC