Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A

Abstract

Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site.

FIGURE 1. Effects of unlabeled eIF5A(Lys), eIF5A(Dhp) and eIF5A(Hpu) on hydroxylation of eIF5A([³H]Dhp)

The reaction was conducted in a typical mixture (see” Experimental Procedures”) using 2 pmol of human eIF5A([³H]Dhp) (33 ng, 0.1 μM) and 0.1 μg of human DOHH. Unlabeled human eIF5A(Lys), eIF5A(Dhp) and eIF5A(Hpu) were added at 0.1, 0.2, 0.5, 1.0 and 2.0 μg and incubation was 45 min at 37° C. The data represent the averages of duplicate experiments.

FIGURE 2. Co-purification of eIF5A(Dhp) with GST-DOHH

A), Detection of co-purified eIF5A by western blot using an eIF5A monoclonal antibody and B), Ponceau S staining of the same membrane (before western) showing the input of close to equal amounts of GST or GST-fusion proteins applied. BL21(DE3) lysates expressing GST or GST fusion proteins of human DOHH (intact enzyme or N- or C- terminal domain) were mixed either with human eIF5A(Lys), eIF5A(Dhp) or eIF5A(Hpu) prior to GSH-Sepharose affinity purification as described under “Experimental Procedures”. The experiment was carried out twice with virtually the same results.

FIGURE 3. Comparison of intact and various truncated eIF5A polypeptides as substrates for DOHH (A–C) and of their binding to GST-DOHH (D)

Clarified lysates of BL21(DE3) cells expressing human eIF5A or eIF5A peptides with a truncation from the N- or C-terminal were used for a DHS assay or for combined DHS/DOHH assay as described under “Experimental Procedures”. A) Fluorogram of SDS gel of a portion of the combined DHS/DOHH reaction mixtures. B) The amounts of radiolabeled hypusine and deoxyhypusine formed in the combined DHS/DOHH reaction, as determined by ion exchange chromatography of acid hydrolysates as described under “Experimental Procedures”. C) Radioactive hypusine as a percent of the total of radioactive hypusine plus deoxyhypusine formed in the combined DHS/DOHH reaction. D) Binding of radiolabeled eIF5A(Dhp) peptides to GST-DOHH from GST-pull down assays. The data (C and D) represent the averages of duplicate experiments.

FIGURE 4. Comparison of substrate binding (A), activities (B) and holoenzyme content (C) of DOHH wild type and mutant enzymes

Groups I and II, mutant enzymes of the His-Glu motifs, group III, those outside of the His-Glu motifs. A) Binding of eIF5A(Dhp) to the GST-DOHH wild type and mutant enzymes was assessed as described under “Experimental Procedures”, using E. coli lysates expressing human GST-DOHH and purified human eIF5A(Dhp). Western blot with eIF5A antibody (top panels) and Ponceau S- stained membrane (before exposure to antibody) to show the amounts of GST-DOHH applied (bottom panels). B) The activities of purified human DOHH mutant enzymes (after cleavage of GST) were determined using radiolabeled eIF5A([³H]Dhp), 0.1 and 0.01 μg of enzymes as described under “Experimental, Procedures” and the activities are expressed at two levels 0.1 (light grey bar) and 0.01μg (dark grey bar). The data represent the averages of duplicate experiments. All the mutant enzymes of group I and E57N, E57Q, E208N, E208Q were totally inactive with no detectable activity even at 1.0 μg. C) Purified DOHH enzymes were electrophoresed under non-denaturing conditions (top panels) for detection of the holo- and apoenzyme, and under denaturing conditions (bottom panels). As described previously (24), purified DOHH enzymes (one major band on SDS-PAGE) resolve into the holoenzyme (lower bands) and the diffused apoenzyme (brackets) upon native gel electrophoresis, suggesting a more compact structure of the former than the latter.

SCHEME 1. Proposed model for the binding of eIF5A(Dhp) to DOHH

The active site His-Glu residues important for binding of iron and the substrate protein are numbered. Side chain carboxyl groups of Glu57 and Glu208 are proposed to interact with the amino group(s) of the deoxyhypusine side chain of eIF5A(Dhp). The γ-carboxyl groups of Glu90 and Glu241 may also contribute to the substrate binding by interaction with the deoxyhypusine residue or other basic residues surrounding it. The six residues, His56, His89, Glu90, His207, His240 and Glu241, implicated in iron binding are in blue. Iron atoms are not included in the diagram, since substrate protein binding does not depend on iron binding. This Scheme represents a simplified hypothetical diagram of the DOHH/eIF5A(Dhp) complex, indicating the key residues involved in the binding without specific indication of orientation of the two proteins.