Region of Elongation Factor 1A1 Involved in Substrate Recognition by Legionella pneumophila Glucosyltransferase Lgt1

Abstract

Lgt1 is one of the glucosyltransferases produced by the Gramnegative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide 50 GKGSFKYAWV 59 of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A of eEF1A and is part of the first turn of helix A. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide 50 GKGSFKYAWV 59 with a higher glucosylation rate than fulllength eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBank TM search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment ( 303 GKASFAYAWV 312 ) were glucosylated by Lgt1. NMR studies with the glucosylated eEF1A-derived decapeptide identified an ␣-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.Legionella pneumophila is a Gram-negative bacterium, causing pulmonary infectious disease in humans. This microorganism is able to infect various free-living protozoa in natural environment as well as macrophages, monocytes, and lung epithelial cells during human disease (1, 2). A plethora of virulence factors, which are important for intracellular proliferation of the bacteria in target eukaryotic cells, and a type IVB secretion system for intracytoplasmic delivery of these effectors have been identified (3). Among the best studied Legionella products are RalF (4) and DrrA (5), which act as exchange factors for Arf1 and Rab1 small GTPases, respectively. Additionally, DrrA has been shown to possess activity of a guanine nucleotide dissociation inhibitor-displacement factor (6). These two proteins were suggested to participate in recruitment of endosomal vesicles and construction of a replicative phagosome, which is a characteristic intracellular niche of Legionella and prerequisite for subsequent proliferation of the bacteria in host cells (7). However, despite considerable progress, many aspects of intracellular biology of L. pneumophila, in particular those apart from processes associated with alterations in vesicular trafficking, remain poorly understood.In our previous investigations we identified three proteins in L. pneumophila (Lgt1, Lgt2, and Lgt3), which possess enzymatic activity and modify eukaryotic elongation factor eEF1A 3 at serine 53 by mono-O-glucosylation (8 -10). This modification inhibits protein synthesis and is eventually lethal to target cells. Expression of Lgt1 is strongly increased during late phase of bacterial growth ...

Elongation Factor 1A Is the Target of Growth Inhibition in Yeast Caused by Legionella pneumophila Glucosyltransferase Lgt1

“…For mutagenesis experiments, a portion of TEF1, containing the coding sequence of the recognized by Lgt1 decapeptide Gly 50 -Val 59 (10), was cut from p572 (pRS-TEF1) with SacI/ClaI and was ligated into pBC, resulting in plasmid p574 (supplemental Table S2). QuikChange mutagenesis of p574 was employed to generate the following substitutions in Tef1: G50A (p644), S53A (p575), S53E (p688), S53C (p686), S53K (p687), F54A (p579), K55A (p645), Y56A (p580), W58A (p581), and V59A (p668) (supplemental Table S2; for primers used, see supplemental Table S3).…”

“…Purification of His-tagged Hbs1 from E. coli and His-tagged Tef1 (eEF1A) from S. cerevisiae was described previously (10,11). Wild type and mutant versions of His-tagged Lgt1 were expressed from plasmids p558, p562, p563, and p578 (supplemental Table S2) in E. coli BL21(DE3).…”

“…Lgt1 monoglucosylates eEF1A on serine 53 and yeast Hbs1 on serine 213 (10). These target serine residues are located within conserved regions, exhibiting significant homology between the two proteins.…”

“…Thus far, it is not clear whether the lethal effect of Lgt1 is linked solely to modification of eEF1A or glucosylation of Hbs1 also contributes to toxic effects caused by the Legionella enzyme. Moreover, it was demonstrated recently that a short peptide encompassing region of the elongation factor from glycine 50 to valine 59 was effectively recognized and glucosylated by Lgt1 (10 yet identified eukaryotic protein(s), possessing homologous sequences can be also substrate(s) of Legionella glucosyltransferases and participate in intoxication mechanisms. In contrast, natural target selection of a region encompassing serine 53 in eEF1A for glucosylation by a microbial effector toxin suggests importance of this area for biological functions of the elongation factor.…”

“…The crystal structure of Lgt1 has been solved, allowing the characterization of the Legionella enzyme as a GT-A glucosyltransferase structurally related to clostridial glucosylating toxins (8,9). Known substrates of Lgts are eukaryotic elongation factor 1A (eEF1A) 3 and eRF3 (eukaryotic release factor 3)-related protein Hbs1 (Hsp70 subfamily B suppressor 1) (10).…”