eIF5A is unique in that it is the only cellular protein activated by hypusine modification. eIF5A consists of two β-sheet core domains, a basic N-terminal domain with an exposed hypusine site loop and an acidic C-terminal domain (). The high sequence and structural conservation of eIF5A may be dictated by structural requirements for its interaction with the hypusine modification enzymes, eIF5A downstream effectors or both. We undertook a comprehensive mutagenesis study of human eIF5A-1 to dissect the structural elements of this protein required for its biological activity and for its hypusine modification and thereby to gain insights into its function. In spite of the high sequence conservation of eIF5A, it was remarkably resilient to individual alanine substitutions and a majority of mutant proteins actively supported S. cerevisiae growth. We have identified only a very few amino acid residues in the exposed hypusine loop as the critical sites for its activity ( and ). Our results confirm the absolute requirement for the deoxyhypusine/hypusine modification in eIF5A function and provide new evidence that the hypusine loop is critical for its interaction with downstream effector molecules and that the β-sheet core structures of both the N- and C-terminal domains of eIF5A (, aa17–82 and aa85–146) are essential for eIF5A biological function.
Summary of characteristics of heIF5A mutant proteins.
The loss of eIF5A function in various mutants may result from their defects in effector binding, instability and/or inability to be modified by DHS. Careful analysis of properties of the selected eIF5A mutant proteins, summarized in , revealed distinct sequence requirements for its growth supporting activity and as substrates for DHS and DOHH. We have identified Lys47, Gly49, Dhp/Hpu50, Gly52 and Lys55 as those residues vitally important for the biological activity of eIF5A in supporting yeast growth (). The absolute requirement of the deoxyhypusine/hypusine residue is confirmed by the total lack of activity of Lys50 site mutants, including K50A, K50D, K50I and K50R (regardless of substituting amino acid), when these mutant proteins are stably expressed (). Beside Lys50, the two neighboring Gly residues, Gly49 and Gly52 are probably most critical in eIF5A activity. No growth support was observed, even though both mutant proteins G49A and G52A are stable in S. cerevisiae () and are modified by DHS and DOHH, albeit at reduced efficiency. Being adjacent to Lys50, these two Gly residues are likely to be critical for the β-turn structure of -Gly-X-Y-Gly- motif, the proper orientation of deoxyhypusine/hypusine side chain and the precise configuration of the hypusine loop in its binding to effectors. K55A human mutant is totally inactive in supporting growth, although Lys55 is located adjacent to the hypusine loop (aa46–54, ). In an independent yeast eIF5A mutation study, the yeast K56A, a counterpart of human K55A, is modified by yeast DHS and supports S. cerevisiae growth at 25 °C (Dias et. al., Unpublished results), however it does exhibit a temperature sensitive growth phenotype at 37 °C, in spite of its stability. Judging from this temperature sensitive phenotype of yeast K56A and its location at the base of the hypusine loop, Lys55 is not likely to be centrally involved in effector binding. Instead, it may indirectly affect the proper orientation of the hypusine loop. Comparison of the three different heIF5A mutants, K47A, K47D and K47R, provides an interesting clue on the role of Lys47. The three mutant proteins are all effectively modified by DHS, yet widely differ in their growth supporting activity. K47R supports growth as well as the wild type protein, and K47A does at a reduced rate. In contrast, no growth is observed upon expression of the K47D mutant. Therefore, the basic residue at 47 is predicted to be involved in an ionic interaction with an acidic effector adaptor site. Two other mutants, H51A and P74A, supported growth but at much reduced rates (), suggesting that these proteins are not functioning optimally. Since they are apparently stable and are effectively modified by DHS, alanine substitution of His51 or Pro74 may adversely affect effector binding.
Since the amino acid sequence surrounding the hypusine residue (STSK
) is very basic and hydrophilic, this loop may interact with specific nucleotide sequences of RNA (52
), acidic proteins or ribonucleoprotein complexes. The β
-sheet structure of the C-terminal domain of eIF5A resembles an oligonucleotide binding fold and has also been implicated in RNA binding. This C-terminal domain contains a stretch of highly conserved hydrophobic amino acids (89-F
-102) that was proposed as a potential effector domain involved in protein-protein interaction (53
). Indeed, alanine substitution of Leu91 or Leu101 caused a reduction in growth rate. Analyzing the position of both amino acids in the human eIF5A-1 model, Leu91 and Leu101 are localized at the hydrophobic core of the β-barrel (). Substitution of either of the two leucines by alanine could easily disrupt the tertiary structure. Without a properly folded β-barrel, the mutant proteins are likely more sensitive to to proteolytic degradation as evidenced from their reduced level in HHY212d strains and in BL21(DE3) lysates. Moreover, they may lose important interactions with downstream effectors. The growth defect of L101A may be largely due to instability of the mutant protein, since its level is drastically reduced (). Furthermore, the yeast L102A counterpart exhibits a temperature sensitive phenotype, being unstable only at the non-permissive temperature (29
). Since the growth defect is observed even at 25–30 C for the human L101A mutation, the severity of destabilization differs in the two species.
Although DHS and DOHH enzymes are totally specific for eIF5A and likely recognize the β-sheet core structure of eIF5A N-terminal domain (45
), specific sequence requirements for their substrates were unknown. As for the DHS reaction, in addition to the key residue Lys50, Gly52 and Lys55 seem important for DHS reaction, judging from markedly reduced efficiency of deoxyhypusine synthesis in G52A and K55A mutant proteins. The charge at residue 47 of eIF5A apparently does not matter, since all three mutants, K47A, K47D and K47R are excellent substrates for DHS (). However, in contrast to DHS, DOHH discriminates against K47D, while effectively hydroxylating K47A and K47R. Another residue, His51, also is important for the DOHH, but not for the DHS reaction, since alanine substitution at this site blocks hydroxylation without affecting deoxyhypusine synthesis. Our recent eIF5A/DOHH binding study revealed that the four conserved glutamic acid residues of the DOHH active site are critical for substrate binding (45
), anchoring the deoxyhypusine side chain of eIF5A(Dhp) and possibly other basic residues in the vicinity. Thus, neighboring basic residues, such as Lys47 and His51 of the eIF5A substrate, may also contribute to its binding to DOHH through ionic interaction with the enzyme’s active site glutamic acids.
The role of eIF5A and its hypusine modification in translation has been a longstanding mystery. eIF5A enhances methionyl-puromycin synthesis in a deoxyhypusine/hypusine-dependent manner in vitro
). Recently, it was shown that eIF5A binds to actively translating ribosomes and that conditional mutants of eIF5A are super-sensitive to protein synthesis inhibitors (20
). The aIF5A structure is partially superimposable on its bacterial ortholog, elongation factor P (EF-P) which contains a third domain and resembles the structure of tRNA. Whether there is an actual functional mimicry, over ribosome association, to accompany the tRNA-related structures of eIF5A/aIF5A/EF-P will require further experimentation. Addition of modified eIF5A (eIF5A(Dhp)) to an eIF5A-depleted lysate of UBHY-R strain enhances total protein synthesis by two-fold (CA Henderson and JWB Hershey, unpublished results) in vitro,
whereas no enhancement is observed with unmodified eIF5A precursor. Expression of human eIF5A (wild type or K47R) in the UBHY-R strain restores protein synthesis in vivo
as well as growth. All these findings are consistent with a role of eIF5A in translation. However, rapid eIF5A depletion did not result in complete inhibition of protein synthesis in vitro
or in vivo
. While eIF5A is a cellular factor essential for cell proliferation and viability, there seems to be no absolute requirement for it for global protein synthesis. Instead, it may be required for optimal and balanced translation of a large number of endogenous mRNAs, especially ones involved in cell-cycle progression. Recently, genes involved in actin polarization, a process necessary for the G1/S transition in yeast, were isolated as high-copy suppressors of temperature-sensitive eIF5A mutants (28
). It remains to be determined whether eIF5A affects the translation of overall mRNAs or a subpopulation of mRNAs that are critical for cell proliferation.
eIF5A activity in vitro and in vivo depends not only on a long and basic deoxyhypusine/hypusine side chain, but also on a specific configuration of the exposed hypusine loop (aa46–54) with Gly-Dhp/Hpu-His-Gly- tip structure properly oriented by neighboring residues such as Lys55 and a basic charge at residue 47. Such a precise structural requirement suggests that this deoxyhypusine/hypusine loop docks into a defined rigid space of its biological effector molecule. It is a great future challenge to identify this binding pocket of eIF5A partner molecules (proteins, RNA or complexes) and to elucidate the mode of eIF5A action.