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Abstract:
Intracellular trafficking of endosomes requires the cooperation of many soluble and membrane-bound components. The small GTPase Rab5 recruits a set of cytosolic effector molecules on the early endosome necessary for endosome biogenesis. Among them is the early endosome-associated autoantigen 1 (EEA1), whose homodimeric architecture consists of an N-terminal C2H2 Zn-finger followed by ~1200 residues of coiled coil, and a C-terminal FYVE domain. EEA1 binding to active Rab5, phosphatidylinositol 3-phosphate (PI3P), and syntaxins 6 and 13, helps regulate endosome tethering and fusion. This study utilized hydrogen deuterium exchange mass spectrometry (HDX-MS) to study the backbone dynamics of EEA1 and how they are regulated by Rab5 binding. Various mutants of EEA1 were generated to modulate coiled-coil stability. This produced valuable insights into how the fine-tuning of the coiled-coil structure controls binding events. We also found allosteric modulation of the coiled-coil structure upon binding Rab5 and noted that this allosteric modulation could be controlled by altering the stability of the coiled-coil.
