Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Abstract

A bacterial inverting glycosyltransferase EarP transfers rhamnose from dTDP-β-l-rhamnose (TDP-Rha) to Arg32 of translation elongation factor P (EF-P) to activate its function. We report here the structural and biochemical characterization of Pseudomonas aeruginosa EarP. In contrast to recently reported Neisseria meningitidis EarP, P. aeruginosa EarP exhibits differential conformational changes upon TDP-Rha and EF-P binding. Sugar donor binding enhances acceptor binding to EarP, as revealed by structural comparison between the apo-, TDP-Rha-, and TDP/EF-P-bound forms and isothermal titration calorimetry experiments. In vitro EF-P rhamnosylation combined with active-site geometry indicates that Asp16 corresponding to Asp20 of N. meningitidis EarP is the catalytic base, whereas Glu272 is another putative catalytic residue. Our study should provide the basis for EarP-targeted inhibitor design against infections from P. aeruginosa and other clinically relevant species. IMPORTANCE Posttranslational rhamnosylation of EF-P plays a key role in Pseudomonas aeruginosa, establishing virulence and antibiotic resistance, as well as survival. The detailed structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP from P. aeruginosa not only demonstrates that sugar donor TDP-Rha binding enhances acceptor EF-P binding to EarP but also should provide valuable information for the structure-guided development of its inhibitors against infections from P. aeruginosa and other EarP-containing pathogens.

Toward a structome of Acinetobacter baumannii drug targets

“…Proline is one of the slowest amino acids to form a peptide bond due to its rigid nature, necessitating elongation factor P (EFP for protein generation. EFP resembles tRNA in structure and bacterial EFP is a three domain structure compared to eukaryotic elongation factor 5A (EIF5A) which is a two domain structure . There is minimal structural resemblance of EIF5A to EFP which would aid in developing specific inhibitors without human toxicity.…”

A β-hairpin epitope as novel structural requirement for protein arginine rhamnosylation

“…Tight binding of the native protein substrate EF-P was measured with a binding constant K d of 473 ± 94 nM for EarP ( Figure 3A), in agreement with reported values. 29 Conversely, binding of 11mer_Pa to EarP could not be detected by ITC using a range of increasing concentrations ( Figure 3B). Even a displacement experiment, in which EF-P was titrated into a solution of pre-formed 11mer_Pa-EarP-TDP complex, did not reveal binding of 11mer-Pa to EarP (data not shown).…”

“…As revealed in various structural studies, [27][28][29] the acceptor binding site of EarP is unusually large and multiple contacts between active site residues of EarP and amino acids of domain I of EF-P are necessary for protein substrate recognition. Upon examining the co-crystal structure of EarP and domain I of EF-P from P. aeruginosa 29 (PDB 6J7M) it is evident that the majority of EF-P residues involved in binding to EarP are located in the hairpin with Arg32 at its tip (Figure 2A). Multiple EarP active site residues are involved in acceptor protein recognition and form both main-chain and side-chain promoted H-bonds, salt bridges, and hydrophobic interactions ( Table S2).…”

“…An anti-Arg Rha antibody has also been developed, allowing for facile (in vitro) monitoring of arginine rhamnosylation. 25,27 Several (co)-crystal structures have been reported for EarP (from P. putida, 27 N. meningitidis 28 and P. aeruginosa 29 ), also in complex with its EF-P substrate, providing insight into the specific amino acid interactions and the catalytic mechanism of rhamnosylation. As only a single Arg residue in EF-P is modified, an important yet unexplored aspect of this novel glycosylation system is the basis for the observed specificity in recognizing this arginine residue.…”

“…27 Domain I, commonly referred to as a "KOWdomain", 27 is a conserved domain in various ribosomeassociated proteins involved in transcription and translation, 30 and it appears to contain all recognition elements necessary to promote Arg rhamnosylation. [27][28][29] A recent study indicates that structural elements are more important than a specific sequon to promote EarP-mediated rhamnosylation. 31 However, the precise determinants for recognition by EarP are currently not known.…”