Abstract
The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288–P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.
Highlights
Hypusine (N-(4-amino-2-hydroxybutyl) lysine), an unusual amino acid, is formed by the post-translational modification of a lysine residue with the addition of the aminobutyl moiety from the polyamine spermidine
The binding of eukaryotic translation factor 5A (eIF5A) to translating ribosomes in a hypusine-dependent manner indicates modifications of the specific binding to the translational machinery [4,5], suggesting the important role of eIF5A receiving hypusine modifications in translation processes, which was supported by evidence that the hypusine modification of eIF5A is vital for the growth and survival of eukaryotes, from yeast to mammals [6,7,8]
We propose that NAD+ binding to PhoDHS induces the stabilization of the flexible loop structure, but that the alternative conformations indicate the action of the loop as a “lid.” In an open conformation, the active site is exposed to receive NAD+, and the lid is closed over NAD+ in conf2
Summary
Hypusine (N-(4-amino-2-hydroxybutyl) lysine), an unusual amino acid, is formed by the post-translational modification of a lysine residue with the addition of the aminobutyl moiety from the polyamine spermidine. The hypusine modification uniquely occurs in eukaryotic translation factor 5A (eIF5A) precursor proteins [1]. EIF5A was originally designed as a translation initial factor, it has been shown that eIF5A is more directly related to elongation rather than the initiation step. The hypusine-modification of eIF5A is involved in the mRNA transportation and translation elongation on the ribosome [2,3]. Previous studies reported that the hypusine residue is essential for the homodimerization of eIF5A and affects its subcellular location [1,2,9,10]
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