Structural and functional analysis of FIP2 binding to the endosome-localised Rab25 GTPase.
Rab small GTPases are the master regulators of intracellular trafficking in eukaryotes. They mediate spatial and temporal recruitment of effector proteins to distinct cellular compartments through GTP-induced changes in their conformation. Despite numerous structural studies, the molecular basis for Rab/effector specificity and subsequent biological activity remains poorly understood. Rab25, also known as Rab11c, which is epithelial-specific, has been heavily implicated in ovarian cancer development and independently appears to act as a tumour suppressor in the context of a distinct subset of carcinomas. Here, we show that Rab25 associates with FIP2 and can recruit this effector protein to endosomal membranes. We report the crystal structure of Rab25 in complex with the C-terminal region of FIP2, which consists of a central dimeric FIP2 coiled-coil that mediates a heterotetrameric Rab25-(FIP2)2-Rab25 complex. Thermodynamic analyses show that, despite a relatively conserved interface, FIP2 binds to Rab25 with an approximate 3-fold weaker affinity than to Rab11a. Reduced affinity is mainly associated with lower enthalpic gains for Rab25:FIP2 complex formation, and can be attributed to subtle differences in the conformations of switch 1 and switch 2. These cellular, structural and thermodynamic studies provide insight into the Rab11/Rab25 subfamily of small GTPases that regulate endosomal trafficking pathways in eukaryotes.
['Lall|Patrick|P|', 'Horgan|Conor P|CP|', 'Oda|Shunichiro|S|', 'Franklin|Edward|E|', 'Sultana|Azmiri|A|', 'Hanscom|Sara R|SR|', 'McCaffrey|Mary W|MW|', 'Khan|Amir R|AR|']
D018797:Cell Cycle Proteins D018360:Crystallography, X-Ray D011992:Endosomes D006367:HeLa Cells D006801:Humans D026901:Membrane Transport Proteins D011485:Protein Binding D020836:Protein Structure, Quaternary D017434:Protein Structure, Tertiary D021381:Protein Transport D035582:Transcription Factor TFIIIA D020691:rab GTP-Binding Proteins
FIP2 Rab25 Cancer Endosome Crystal structure Rab11 ERC GAP GDI GEF GTP GTP/GDP dissociation inhibitor GTPase-activating protein ITC MBP RBD Rab11 family interacting protein 2 Rab11-binding domain endosomal-recycling compartment guanine nucleotide exchange factor guanosine 5′ triphosphate isothermal titration calorimetry maltose-binding protein