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. 2017 Aug 21;45(14):8392-8402.
doi: 10.1093/nar/gkx532.

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis

Byung-Sik Shin  1 , Takayuki Katoh  2 , Erik Gutierrez  1 , Joo-Ran Kim  1 , Hiroaki Suga  2 , Thomas E Dever  1
Affiliations

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis

Byung-Sik Shin et al. Nucleic Acids Res. .

Abstract

Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline.

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Figures

Figure 1.
Figure 1.
Imino acid proline and not tRNAPro imposes eIF5A requirement for polyproline peptide synthesis. (A) Cloverleaf structures of yeast tRNAPro and tRNAPhe depicting their misacylation by flexizymes eFx and dFx and cyanomethyl ester (CME)-phenylalanine and 3,5-dinitrobenzyl ester (DBE)-proline, respectively. (B) Scheme for in vitro reconstituted translation elongation assay. (C) Maximum fractions (Ymax) of MFFF (lanes 1–4) and MPPPK (lanes 5–8) synthesis in elongation assays (Supplementary Figure S1) performed with the indicated canonical Pro-tRNAPro or Phe-tRNAPhe or with misacylated Pro-tRNAPhe or Phe-tRNAPro in the presence of 1 mM spermidine and in the absence (white bars) or presence (black bars) of 5 μM hypusinated eIF5A. Fold stimulation of Ymax by eIF5A is presented (above bars) for each aminoacyl-tRNA. Error bars are standard deviations (SD) from at least three independent experiments.
Figure 2.
Figure 2.
eIF5A-independent synthesis of homopolymers of azetidine-2-carboxylic acid (AZC), but not of other proline analogs. Maximum fractions of MPPPK peptide synthesis in elongation assays (Supplementary Figure S2) performed with the indicated proline analogs (1, l-proline; 2, α-methyl-l-proline; 3, 3,4-dehydro-dl-proline; 4, cis-4-hydroxy-l-proline; 5, pipecolic acid; 6, l-azetidine-2-carboxylic acid) in the presence of 1 mM spermidine and in the absence (white bars) or presence (black bars) of 5 μM hypusinated eIF5A. (Bottom) Fold stimulation of Ymax by eIF5A was calculated for each reaction in the upper panel (see also Supplementary Figure S2). Error bars are SD from at least three independent experiments (A) and calculated propagated SD (B).
Figure 3.
Figure 3.
Both hypusinated and unhypusinated eIF5A substitute for polyamines to stimulate general translation. (A) Maximum fractions of MFFF peptide synthesis obtained in polyamine-deficient in vitro reconstituted translation assays supplemented with the indicated amounts of spermidine, spermine or putrescine (structures depicted above plots) were plotted and fit to the Michaelis–Menten equation. K1/2(endpoint) is the polyamine concentration at which 50% of the Ymax is obtained. (B, C) Maximum fractions of MFFF (B) and MPPPK (C) peptide synthesis obtained in polyamine-deficient in vitro reconstituted translation assays supplemented with no eIF5A (No), 5 μM wild type hypusinated eIF5A [WT(+Hyp)], or three versions of eIF5A (5 μM) lacking hypusine: wild type eIF5A (K51), eIF5A-K51A, or eIF5A-K51R, as indicated (see also Supplementary Figure S3). Error bars are SD from at least three independent experiments (B, C).
Figure 4.
Figure 4.
Models of polyamine and eIF5A stimulation of general and polyproline peptide synthesis. In the absence of eIF5A polyproline synthesis (left panel) stalls with diproline bound to the P-site tRNA and Pro-tRNA in the A site of an 80S elongating ribosome. Binding of eIF5A (orange) in the E site positions the hypusine residue (green) so that it can interact with the acceptor stem of the P-site tRNA and facilitate peptide bond formation. General (polyPhe) translation (right panel) requires polyamines such as spermidine; however, both unmodified and hypusinated eIF5A can functionally substitute for polyamines to stimulate general translation. The precise binding sites for spermidine on the ribosome are not known; however, given the overlap between eIF5A and polyamine function in general translation, polyamines are proposed to interact with the P-site tRNA to stabilize its binding to the ribosome and enhance its reactivity in peptide bond formation.
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