Hypusination is a two-step enzyme-mediated post-translational modification that converts lysine into hypusine. The modification is unique to the eukaryotic translation factor eIF5A, which is essential for overcoming ribosome stalling in polyproline regions. Dysregulation of hypusination is implicated in cancer, diabetes and hypusination-related neurodegeneration, including the recently identified neurodevelopmental syndrome deoxyhypusine synthase (DHS) disorder. In the first step, deoxyhypusine is generated by DHS via the NAD-dependent transfer of the aminobutyl moiety from spermidine to the lysine side chain of eIF5A. Subsequent hydrolyation by deoxyhypusine hydroxylase generates hypusine. Structures of DHS have yielded mechanistic insights into its active-site composition, spermidine-binding mode and inhibition, whereas eIF5A has been resolved in complex with the translating ribosome; however, there remains no structural information on the complexes key to the hypusination reaction. The lack of mechanistic detail is particularly pressing, given the recent discovery of DHS disorder. To shed light on the mechanism of the rate-limiting step of hypusination, Wątor et al. determined the cryo-electron miscopy structure of the human eIF5A–DHS complex. The structure reveals that one monomer of eIF5A binds to tetrameric DHS and, in concert with hydrogen–deuterium exchange mass spectrometry, highlights how binding of the N-terminal domain of eIF5A unmasks the DHS active site via the movement of a ball-and-chain motif in the N-terminal helix of DHS. Directed mutagenesis of both proteins identified key residues in the interaction interface, which enabled a crystal structure of DHS to be solved in the deoxyhypusination transition state; this clarified the mechanism by which the spermidine-derived 4-aminobutyl moiety is transferred via DHS Lys329, stabilized by the rearrangement of Trp327, to Lys50 of eIF5A. This work provides much needed mechanistic detail on hypusination and reveals how clinically relevant mutations affect this key interaction, providing the basis for future therapeutic development.
Original reference: Nat. Commun. 14, 1698 (2023)
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