Elongation factor P

Elongation factor P (EF-P) is a small (21 kDa) protein which comprises three mainly β-barrel domains (I, II and III, Fig. 3A). Its domain arrangement and the overall shape resemble that of a tRNA, with the N-terminal domain I representing the acceptor end and the C-terminal domain III resembling the anticodon stem of tRNA (Fig. 3D) (Choi & Choe, 2011; Hanawa-Suetsugu et al, 2004). Furthermore, most of the surface is negatively charged (Hanawa-Suetsugu et al, 2004). Comparison of EF-P structures from different organisms and within different asymmetric units indicates that domain I

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can adopt different orientations relative to domain II and III (Choi & Choe, 2011). EF-P is universally conserved in all three domains of life; sequence and structure similarities of EF-P and its archaeal and eukaryotic orthologs called initiation factors 5A (aIF5A and eIF5A, respectively) indicate functional conservation among the bacterial, archaeal and eukaryotic orthologs (Park et al, 2010). Although the archeal and eukaryotic orthologs lack the C-terminal domain III of bacterial EF-P, 42% residues of Thermus thermophilus EF-P are conserved or similar in eIF5As and the structures of the remaining domains superimpose very well (Fig. 3D) (Hanawa-Suetsugu et al. 2004). Whether the discrepancy in domain numbers is the result of a deletion event in eukaryotes or a duplication event in bacteria is not known (Hanawa-Suetsugu et al, 2004).

A C E

I II

III

B D

Fig. 3: Structures of EF-P/eIF5A, tRNA and the ribosome

A) EF-P from E. coli illustrated as cartoon with electrostatic potential of the surface (PDB: 3A5Z). Domain numbering as indicated. B) tRNA^fMet from T. thermophilus (PDB: 3HUY). C) eIF5A from Saccharomyces cerevisiae (PDB: 3ER0). D) Superposition of E. coli EF-P (black, PDB: 3A5Z), eIF5A from S. cerevisiae (purple, PDB: 3ER0) and initiator tRNA from T. thermophilus (grey surface, PDB: 3HUY). E) Ribosome-bound EF-P and initiator tRNA (red and blue, respectively; PDB: 3HUY) aligned onto ribosome-bound A- and P-site tRNAs (light and dark blue, respectively; PDB: 4v5d) from T. thermophilus. Figures were generated in PyMOL (https://www.pymol.org).

Deletion strains in E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens and Salmonella enterica serovar typhimurium suggest that EF-P is not essential in bacteria (Baba et al, 2006; Balibar et al, 2013; Peng et al, 2001; Zou et al, 2011). In yeast and higher eukaryotes eIF5A is essential and occurs in different tissue-specific isoforms (reviewed in (Park et al, 2010)). EF-P binds the ribosome in a 1:1 molar ratio (Aoki et al, 2008). A structural investigation showed that it spans both ribosomal subunits and binds between the E and P site (Fig. 3E) (Blaha et al, 2009). Its C-terminal domain III contains a conserved sequence motif (GDT) in a flexible loop (not resolved in the crystal structure) which was proposed to interact with the 30S ribosome or the mRNA (Choi & Choe, 2011). Its

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terminal domain I - in analogy to the acceptor end of tRNA - points towards the peptidyl transferase center and contacts the CCA end of the P-site tRNA (Blaha et al, 2009). At the very tip of domain I, EF-P and its orthologs are posttranslationally modified at a conserved lysine or arginine residue, notably by different enzymes and with different modifications (Fig. 4). In E. coli EF-P is modified at Lys34 with an R-β-lysine and a hydroxyl-group. The first step of the EF-P lysylation pathway (Fig. 4A) involves the conversion of S-α-Lys to R-β-Lys by a homolog of lysine 2,3 aminomutase (LAM) family named EpmB (YjeK) (Bailly & de Crecy-Lagard, 2010; Behshad et al, 2006; Roy et al, 2011). R-β-Lys is then activated by adenylation and the lysyl moiety is transferred to the ε-amino group of Lys34 of EF-P. This step is catalyzed by EpmA (alternative names: YjeA, PoxA or GenX), a homolog of the catalytic domain of class II Lys-tRNA synthetases (Lys-RS2) (Ambrogelly et al, 2010; Bailly & de Crecy-Lagard, 2010; Navarre et al, 2010; Yanagisawa et al, 2010). A third modifying enzyme EpmC (YfcM) hydroxylates the conserved Lys34, presumably at its C5(δ) (Peil et al, 2012). The modification further extends EF-P into the direction of the peptidyl transferase center and a molecular model suggests that it could reach within 2 Å of the C-terminal amino acid of the P site-bound tRNA (Lassak et al, 2015). Lysylation of EF-P is relevant for its catalytic proficiency in vivo and in vitro (Navarre et al, 2010; Park et al, 2012; Zou et al, 2012).

Fig. 4: Modification of EF-P, e/aIF5A

A) EF-P modification pathway in E. coli. B) Rhamnosylated EF-P from S. oneidensis / P.

aeruginosa. C) Hypusinylated a/eIF5A.

Modifications are depicted in boxes. Figure adapted from (Doerfel & Rodnina, 2013)

Notably, ~70% of bacteria do not encode EpmA or EpmB, suggesting that either EF-P remains unmodified or is modified by different enzymes in these organisms (Bailly & de Crecy-Lagard, 2010).

Indeed, recent bioinformatics studies and biochemical data indicate that a wide range of EF-P modifications have evolved among different organisms: For example, Shewanella oneidensis and P.

aeruginosa belong to a subclass of bacteria (~9%) with a strictly conserved Arg and do not encode EpmA, B, C but the glycosyltransferase EarP (Lassak et al, 2015; Rajkovic et al, 2015). In both

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organisms EF-P is rhamnosylated by EarP at Arg32/34 (Fig. 4B), a position structurally equivalent to Lys34 of E. coli EF-P. Deletion of earP in S. oneidensis phenocopys strains where EF-P cannot be modified, indicating that, similar to the lysylation of E. coli EF-P, glycolysation is required to activate EF-P in these organisms (Lassak et al, 2015; Rajkovic et al, 2015). Furthermore, several bacterial species encode a gene cognate to deoxyhypusine synthase (DHS) which modifies eIF5A in eukaryotes (Brochier et al, 2004). In eukaryotes eIF5A Lys51 is transformed into hypusine [N^ε -(4-aminobutyl-2-hydroxy)-l-lysine] (Fig. 4C) by addition of a spermidine moiety by DHS and subsequent hydroxylation of the deoxyhypusine intermediate by deoxyhypusine hydroxylase (DOHH) (Park, 2006). In archaea aIF5A exists in both hypusinated and deoxyhypusinated forms (Park et al, 2010). While the modification in eIF5A is essential in eukaryotes (Park et al, 2010; Schnier et al, 1991), the deletion of EF-P or of the EF-P-modifying enzymes EpmA or EpmB but not EpmC in bacteria results in pleiotropic phenotypes reducing the general fitness: growth defects (Abratt et al, 1998; Balibar et al, 2013;

Charles & Nester, 1993; Iannino et al, 2012; Kaniga et al, 1998; Peng et al, 2001), changed susceptibility to a wide range of external stressors such as antibiotics (Abratt et al, 1998; Balibar et al, 2013; Bearson et al, 2011; Iannino et al, 2012; Navarre et al, 2010; Zou et al, 2012), motility defects (Bearson et al, 2011) and the reduction of the virulence potential (Charles & Nester, 1993; Iannino et al, 2012; Kaniga et al, 1998; Marman et al, 2014; Navarre et al, 2010; Peng et al, 2001) observed in a great range of organisms (E. coli, P. aeruginosa, S. typhimurium, Bacillus subtilis, A. tumefaciens, Shigella flexneri and Brucella abortus).

EF-P was identified in 1975 as a protein which increases the yield of formylmethionyl-puromycin (fMet-Pmn) (Glick & Ganoza, 1975). In the following, EF-P was shown to stimulate poly-Phe/Lys synthesis and the translation of a natural mRNA (Aoki et al, 1997; Aoki et al, 2008; Ganoza & Aoki, 2000; Ganoza et al, 1985; Glick & Ganoza, 1975; Glick & Ganoza, 1976; Green et al, 1985). However, the identified effects were relatively small (up to 2-fold) and their cellular relevance remained unclear. Based on biochemical and structural investigations, EF-P was proposed to position the tRNA^fMet in the P site (Aoki et al, 2008; Blaha et al, 2009) or to promote the first peptide bond (Blaha et al, 2009; Glick & Ganoza, 1975). For a/eIF5A serveral functions have been proposed, e.g. to promote the formation of the first peptide bond, the translation of certain mRNAs, to affect peptide release and to influence cell-cycle progression as well as mRNA decay (reviewed in (Zanelli et al, 2006)). Inactivation of eIF5A leads to accumulation of polysomes and increased ribosome transit times, which indicates that the factor is involved in translation elongation (Gregio et al, 2009; Saini et al, 2009). However, the cellular concentration of EF-P in the cell (1/10 of ribosomes) (An et al, 1980) suggests that its function is not required in general but is restricted to a specific translational event (Saini et al, 2009) which remained unknown.

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