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• W2019410617 abstract "Lgt1 is one of the glucosyltransferases produced by the Gram-negative 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 50GKGSFKYAWV59 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 50GKGSFKYAWV59 with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBankTM 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 (303GKASFAYAWV312) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an α-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase. Lgt1 is one of the glucosyltransferases produced by the Gram-negative 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 50GKGSFKYAWV59 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 50GKGSFKYAWV59 with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBankTM 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 (303GKASFAYAWV312) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an α-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 (1Bitar D.M. Molmeret M. Abu Kwait Y. Int. J. Med. Microbiol. 2004; 293: 519-527Crossref PubMed Scopus (27) Google Scholar, 2Fields B.S. Benson R.F. Besser R.E. Clin. Microbiol. Rev. 2002; 15: 506-526Crossref PubMed Scopus (1265) Google Scholar). 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 (3Juhas M. Crook D.W. Hood D.W. Cell Microbiol. 2008; 10: 2377-2386Crossref PubMed Scopus (170) Google Scholar). Among the best studied Legionella products are RalF (4Nagai H. Kagan J.C. Zhu X. Kahn R.A. Roy C.R. Science. 2002; 295: 679-682Crossref PubMed Scopus (445) Google Scholar) and DrrA (5Murata T. Delprato A. Ingmundson A. Toomre D.K. Lambright D.G. Roy C.R. Nat. Cell Biol. 2006; 8: 971-977Crossref PubMed Scopus (286) Google Scholar), 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 (6Ingmundson A. Delprato A. Lambright D.G. Roy C.R. Nature. 2007; 450: 365-369Crossref PubMed Scopus (265) Google Scholar). 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 (7Ninio S. Roy C.R. Trends Microbiol. 2007; 15: 372-380Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). 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 3The abbreviations used are: eEF1Aeukaryotic elongation factor 1AGSTglutathione S-transferaseTOCSYtotal correlation spectroscopyHbs1heat shock protein 70 subfamily B suppressor 1DOSYdiffusion-ordered spectroscopy. 3The abbreviations used are: eEF1Aeukaryotic elongation factor 1AGSTglutathione S-transferaseTOCSYtotal correlation spectroscopyHbs1heat shock protein 70 subfamily B suppressor 1DOSYdiffusion-ordered spectroscopy. at serine 53 by mono-O-glucosylation (8Belyi I. Popoff M.R. Cianciotto N.P. Infect. Immun. 2003; 71: 181-186Crossref PubMed Scopus (29) Google Scholar, 9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar, 10Belyi Y. Tabakova I. Stahl M. Aktories K. J. Bacteriol. 2008; 190: 3026-3035Crossref PubMed Scopus (106) Google Scholar). This modification inhibits protein synthesis and is eventually lethal to target cells. Expression of Lgt1 is strongly increased during late phase of bacterial growth in broth medium and in Acanthamoeba castellanii (10Belyi Y. Tabakova I. Stahl M. Aktories K. J. Bacteriol. 2008; 190: 3026-3035Crossref PubMed Scopus (106) Google Scholar). Because bacteria taken at the stationary phase of growth are known to possess maximal pathogenic potential (11Byrne B. Swanson M.S. Infect. Immun. 1998; 66: 3029-3034Crossref PubMed Google Scholar, 12Hammer B.K. Swanson M.S. Mol. Microbiol. 1999; 33: 721-731Crossref PubMed Scopus (213) Google Scholar), up-regulation of the glucosyltransferase has been suggested to be involved in virulence of L. pneumophila. Here we studied the recognition of eEF1A1 by Lgt1 and identified the type of glucosylation catalyzed by the enzyme.DISCUSSIONElongation factor eEF1A, with two structurally highly similar isoforms eEF1A1 and eEF1A2, is a large GTP-binding protein, consisting of the N-terminal GTPase domain and two additional domains of β-barrel structure (26Andersen G.R. Nyborg J. Cold Spring Harb. Symp. Quant. Biol. 2001; 66: 425-437Crossref PubMed Scopus (15) Google Scholar). L. pneumophila glucosyltransferase Lgt1 glucosylates eEF1A at serine 53, which is located in the GTPase domain of the elongation factor (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). Here we studied the substrate recognition of eEF1A by Lgt1. Our studies show that domains 2 and 3 of eEF1A are fully dispensable for glucosylation by Lgt1. Moreover, efficient glucosylation by Lgt1 was observed with the decapeptide 50GKGSFKYAWV59 as a substrate. This suggests that the three-dimensional structure of eEF1A is not important for recognition by Lgt1.Many bacterial protein toxins are characterized by extremely high substrate specificity. These examples include C. difficile toxins A and B, which are at least distantly related to Lgt1 (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar), as well as other members of the glucosylating clostridial toxin family. They modify small GTPases of the Rho and/or Ras family in a highly specific manner, depending on the intact folding of the whole substrate protein (27Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). For example, change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin of C. sordellii. Similarly, substitution of phenylalanine 85 in RhoD with serine allowed its glucosylation by toxin B of C. difficile (28Jank T. Pack U. Giesemann T. Schmidt G. Aktories K. J. Biol. Chem. 2006; 281: 19527-19535Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In contrast, the cytotoxic necrotizing factors CNF of E. coli needs only a rather small Rho peptide of 20 residues to catalyze deamidation of the target glutamine residue (29Lerm M. Schmidt G. Goehring U.M. Schirmer J. Aktories K. J. Biol. Chem. 1999; 274: 28999-29004Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Even smaller is the minimum peptide of eEF1A, which is modified by Lgt1. It contains the sequence 50GKGSFKYAWV59 and forms the loop, which is a part of the helix-loop-helix structure of helices A* and A′ of eEF1A. Whereas glycine 50 is the first residue of this loop, the loop ends with lysine 55. Tyrosine 56 and the other residues of the peptide form the first turn of helix A′. Serine 53 is located in the middle of the loop and apparently is freely accessible at the surface of eEF1A.Even smaller truncations (e.g. KGSFKYAWV, GKGSFKYAW, and KGSFKYAW) were substrates for Lgt1, although glucosylation was less efficient. As one can deduce from the experiments, the COOH-terminal part of the peptide was more important for its substrate activity than the NH2-terminal part. In line with this notion were results of mutagenesis studies. Substitution of amino acid residues located COOH-terminally (e.g. lysine 55, tyrosine 56, alanine 57, or tryptophan 58) caused major reduction in glucosylation and substitution of phenylalanine 54 completely prevented glucosylation of the neighboring serine 53.Glucosylation of serine 53 of eEF1A inhibits protein synthesis (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). However, the functional consequences of the modification of serine 53 are not clear on the molecular level. Serine 53, which is glucosylated by Lgt1 in eEF1A, is not present in prokaryotic EF1A (known as EF-Tu) but conserved in many eukaryotic elongation factors 1A. Whereas glucosylation of the small GTPase RhoA by clostridial glucosyltransferases modifies threonine 37, which is essential for nucleotide binding and coordination of magnesium (30Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar), the glucosylation site (serine 53) in eEF1A is not directly involved in nucleotide binding. Glucosylation of RhoA and Ras by clostridial toxins inhibits the active conformation of the protein. The exposed location of serine 53 in eEF1A rather suggests that glucosylation of the elongation factor interferes with binding and/or interaction with other proteins or factors, eventually resulting in inhibition of protein synthesis.Interestingly, the rate of glucosylation of peptide 50GKGSFKYAWV59 was at least 20-fold higher than the glucosylation of a native full-length eEF1A or its recombinant G domain. This suggests that a specific conformation of eEF1A might be the preferred substrate of Lgt1. However, our attempts to find out conditions that cause a major increase in glucosylation by Lgt1 have been unsuccessful so far.The short peptide, which was recognized by Lgt1 as a substrate for glucosylation, prompted us to search for other proteins, harboring this sequence. A BLAST search identified Hsp70 subfamily B suppressor 1 (Hbs1) as a putative target for glucosylation. Recombinant Hbs1 was readily modified by Lgt1-induced glucosylation. Initially Hbs1 was identified in yeast screens as a 70-kDa heat shock cognate protein, which suppressed defects caused by a proteasome mutation in Saccharomyces cerevisiae (31Ohba M. FEBS Lett. 1994; 351: 263-266Crossref PubMed Scopus (25) Google Scholar). Later it was shown that Hbs1 possesses strong homology to release factor eRF3 (32Wallrapp C. Verrier S.B. Zhouravleva G. Philippe H. Philippe M. Gress T.M. Jean-Jean O. FEBS Lett. 1998; 440: 387-392Crossref PubMed Scopus (37) Google Scholar). Hbs1 and Hbs1-interacting eRF1-related factor Dom34 appear to be important for efficient protein synthesis in and growth of yeast cells under conditions of limiting translation initiation (33Carr-Schmid A. Pfund C. Craig E.A. Kinzy T.G. Mol. Cell. Biol. 2002; 22: 2564-2574Crossref PubMed Scopus (57) Google Scholar). In more recent reports, relationships between Hbs1 function and ribosomal mRNA translation were described (34Doma M.K. Parker R. Nature. 2006; 440: 561-564Crossref PubMed Scopus (479) Google Scholar, 35Graille M. Chaillet M. van Tilbeurgh H. J. Biol. Chem. 2008; 283: 7145-7154Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Lee H.H. Kim Y.S. Kim K.H. Heo I. Kim S.K. Kim O. Kim H.K. Yoon J.Y. Kim H.S. Kim do.J. Lee S.J. Yoon H.J. Kim S.J. Lee B.G. Song H.K. Kim V.N. Park C.M. Suh S.W. Mol. Cell. 2007; 27: 938-950Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). At present we have no experimental confirmation of the role of glucosylation of Hbs1 in Legionella intracellular biology or disease. However, it should be noted that both identified targets of Lgt1, namely eEF1A and Hbs1, represent important components of protein synthesis machinery in eukaryotic cells. In our previous studies we demonstrated that the glucosylation of eEF1A takes place following intoxication of EBL cells by Lgt1 (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). However we cannot exclude that modification of Hbs1 is also of importance for Lgt1-induced biological effects (e.g. inhibition of protein synthesis).The identification of a small peptide that is glucosylated by Lgt1 allowed us to study the stereochemical structure of the O-glycosidic bond formed. The anomeric configuration of the product of glucosylation can be either retained or inverted with respect to the donor co-substrate (37Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Annu. Rev. Biochem. 2008; 77: 521-555Crossref PubMed Scopus (1276) Google Scholar). Here we show by NMR spectroscopy that Lgt1 belongs to the retaining type of glucosyltransferases. Therefore, Lgt1 is similar to the clostridial glucosylating toxins, which glucosylate Rho and/or Ras GTPases in a retaining reaction (27Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 38Geyer M. Wilde C. Selzer J. Aktories K. Kalbitzer H.R. Biochemistry. 2003; 42: 11951-11959Crossref PubMed Scopus (32) Google Scholar, 39Vetter I.R. Hofmann F. Wohlgemuth S. Herrmann C. Just I. J. Mol. Biol. 2000; 301: 1091-1095Crossref PubMed Scopus (56) Google Scholar, 40Ziegler M.O. Jank T. Aktories K. Schulz G.E. J. Mol. Biol. 2008; 377: 1346-1356Crossref PubMed Scopus (49) Google Scholar). Sequence comparison of Lgt1 with clostridial toxins have shown some sequence similarity in the surrounding of the conserved DXD motif, which is involved in coordination of manganese ions and binding of the activated co-substrate UDP-glucose. It remains to be studied whether the structure of the catalytic domain of Lgt1 is related to clostridial glucosyltransferases. 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 (1Bitar D.M. Molmeret M. Abu Kwait Y. Int. J. Med. Microbiol. 2004; 293: 519-527Crossref PubMed Scopus (27) Google Scholar, 2Fields B.S. Benson R.F. Besser R.E. Clin. Microbiol. Rev. 2002; 15: 506-526Crossref PubMed Scopus (1265) Google Scholar). 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 (3Juhas M. Crook D.W. Hood D.W. Cell Microbiol. 2008; 10: 2377-2386Crossref PubMed Scopus (170) Google Scholar). Among the best studied Legionella products are RalF (4Nagai H. Kagan J.C. Zhu X. Kahn R.A. Roy C.R. Science. 2002; 295: 679-682Crossref PubMed Scopus (445) Google Scholar) and DrrA (5Murata T. Delprato A. Ingmundson A. Toomre D.K. Lambright D.G. Roy C.R. Nat. Cell Biol. 2006; 8: 971-977Crossref PubMed Scopus (286) Google Scholar), 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 (6Ingmundson A. Delprato A. Lambright D.G. Roy C.R. Nature. 2007; 450: 365-369Crossref PubMed Scopus (265) Google Scholar). 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 (7Ninio S. Roy C.R. Trends Microbiol. 2007; 15: 372-380Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). 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 3The abbreviations used are: eEF1Aeukaryotic elongation factor 1AGSTglutathione S-transferaseTOCSYtotal correlation spectroscopyHbs1heat shock protein 70 subfamily B suppressor 1DOSYdiffusion-ordered spectroscopy. 3The abbreviations used are: eEF1Aeukaryotic elongation factor 1AGSTglutathione S-transferaseTOCSYtotal correlation spectroscopyHbs1heat shock protein 70 subfamily B suppressor 1DOSYdiffusion-ordered spectroscopy. at serine 53 by mono-O-glucosylation (8Belyi I. Popoff M.R. Cianciotto N.P. Infect. Immun. 2003; 71: 181-186Crossref PubMed Scopus (29) Google Scholar, 9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar, 10Belyi Y. Tabakova I. Stahl M. Aktories K. J. Bacteriol. 2008; 190: 3026-3035Crossref PubMed Scopus (106) Google Scholar). This modification inhibits protein synthesis and is eventually lethal to target cells. Expression of Lgt1 is strongly increased during late phase of bacterial growth in broth medium and in Acanthamoeba castellanii (10Belyi Y. Tabakova I. Stahl M. Aktories K. J. Bacteriol. 2008; 190: 3026-3035Crossref PubMed Scopus (106) Google Scholar). Because bacteria taken at the stationary phase of growth are known to possess maximal pathogenic potential (11Byrne B. Swanson M.S. Infect. Immun. 1998; 66: 3029-3034Crossref PubMed Google Scholar, 12Hammer B.K. Swanson M.S. Mol. Microbiol. 1999; 33: 721-731Crossref PubMed Scopus (213) Google Scholar), up-regulation of the glucosyltransferase has been suggested to be involved in virulence of L. pneumophila. Here we studied the recognition of eEF1A1 by Lgt1 and identified the type of glucosylation catalyzed by the enzyme. eukaryotic elongation factor 1A glutathione S-transferase total correlation spectroscopy heat shock protein 70 subfamily B suppressor 1 diffusion-ordered spectroscopy. eukaryotic elongation factor 1A glutathione S-transferase total correlation spectroscopy heat shock protein 70 subfamily B suppressor 1 diffusion-ordered spectroscopy. DISCUSSIONElongation factor eEF1A, with two structurally highly similar isoforms eEF1A1 and eEF1A2, is a large GTP-binding protein, consisting of the N-terminal GTPase domain and two additional domains of β-barrel structure (26Andersen G.R. Nyborg J. Cold Spring Harb. Symp. Quant. Biol. 2001; 66: 425-437Crossref PubMed Scopus (15) Google Scholar). L. pneumophila glucosyltransferase Lgt1 glucosylates eEF1A at serine 53, which is located in the GTPase domain of the elongation factor (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). Here we studied the substrate recognition of eEF1A by Lgt1. Our studies show that domains 2 and 3 of eEF1A are fully dispensable for glucosylation by Lgt1. Moreover, efficient glucosylation by Lgt1 was observed with the decapeptide 50GKGSFKYAWV59 as a substrate. This suggests that the three-dimensional structure of eEF1A is not important for recognition by Lgt1.Many bacterial protein toxins are characterized by extremely high substrate specificity. These examples include C. difficile toxins A and B, which are at least distantly related to Lgt1 (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar), as well as other members of the glucosylating clostridial toxin family. They modify small GTPases of the Rho and/or Ras family in a highly specific manner, depending on the intact folding of the whole substrate protein (27Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). For example, change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin of C. sordellii. Similarly, substitution of phenylalanine 85 in RhoD with serine allowed its glucosylation by toxin B of C. difficile (28Jank T. Pack U. Giesemann T. Schmidt G. Aktories K. J. Biol. Chem. 2006; 281: 19527-19535Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In contrast, the cytotoxic necrotizing factors CNF of E. coli needs only a rather small Rho peptide of 20 residues to catalyze deamidation of the target glutamine residue (29Lerm M. Schmidt G. Goehring U.M. Schirmer J. Aktories K. J. Biol. Chem. 1999; 274: 28999-29004Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Even smaller is the minimum peptide of eEF1A, which is modified by Lgt1. It contains the sequence 50GKGSFKYAWV59 and forms the loop, which is a part of the helix-loop-helix structure of helices A* and A′ of eEF1A. Whereas glycine 50 is the first residue of this loop, the loop ends with lysine 55. Tyrosine 56 and the other residues of the peptide form the first turn of helix A′. Serine 53 is located in the middle of the loop and apparently is freely accessible at the surface of eEF1A.Even smaller truncations (e.g. KGSFKYAWV, GKGSFKYAW, and KGSFKYAW) were substrates for Lgt1, although glucosylation was less efficient. As one can deduce from the experiments, the COOH-terminal part of the peptide was more important for its substrate activity than the NH2-terminal part. In line with this notion were results of mutagenesis studies. Substitution of amino acid residues located COOH-terminally (e.g. lysine 55, tyrosine 56, alanine 57, or tryptophan 58) caused major reduction in glucosylation and substitution of phenylalanine 54 completely prevented glucosylation of the neighboring serine 53.Glucosylation of serine 53 of eEF1A inhibits protein synthesis (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). However, the functional consequences of the modification of serine 53 are not clear on the molecular level. Serine 53, which is glucosylated by Lgt1 in eEF1A, is not present in prokaryotic EF1A (known as EF-Tu) but conserved in many eukaryotic elongation factors 1A. Whereas glucosylation of the small GTPase RhoA by clostridial glucosyltransferases modifies threonine 37, which is essential for nucleotide binding and coordination of magnesium (30Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar), the glucosylation site (serine 53) in eEF1A is not directly involved in nucleotide binding. Glucosylation of RhoA and Ras by clostridial toxins inhibits the active conformation of the protein. The exposed location of serine 53 in eEF1A rather suggests that glucosylation of the elongation factor interferes with binding and/or interaction with other proteins or factors, eventually resulting in inhibition of protein synthesis.Interestingly, the rate of glucosylation of peptide 50GKGSFKYAWV59 was at least 20-fold higher than the glucosylation of a native full-length eEF1A or its recombinant G domain. This suggests that a specific conformation of eEF1A might be the preferred substrate of Lgt1. However, our attempts to find out conditions that cause a major increase in glucosylation by Lgt1 have been unsuccessful so far.The short peptide, which was recognized by Lgt1 as a substrate for glucosylation, prompted us to search for other proteins, harboring this sequence. A BLAST search identified Hsp70 subfamily B suppressor 1 (Hbs1) as a putative target for glucosylation. Recombinant Hbs1 was readily modified by Lgt1-induced glucosylation. Initially Hbs1 was identified in yeast screens as a 70-kDa heat shock cognate protein, which suppressed defects caused by a proteasome mutation in Saccharomyces cerevisiae (31Ohba M. FEBS Lett. 1994; 351: 263-266Crossref PubMed Scopus (25) Google Scholar). Later it was shown that Hbs1 possesses strong homology to release factor eRF3 (32Wallrapp C. Verrier S.B. Zhouravleva G. Philippe H. Philippe M. Gress T.M. Jean-Jean O. FEBS Lett. 1998; 440: 387-392Crossref PubMed Scopus (37) Google Scholar). Hbs1 and Hbs1-interacting eRF1-related factor Dom34 appear to be important for efficient protein synthesis in and growth of yeast cells under conditions of limiting translation initiation (33Carr-Schmid A. Pfund C. Craig E.A. Kinzy T.G. Mol. Cell. Biol. 2002; 22: 2564-2574Crossref PubMed Scopus (57) Google Scholar). In more recent reports, relationships between Hbs1 function and ribosomal mRNA translation were described (34Doma M.K. Parker R. Nature. 2006; 440: 561-564Crossref PubMed Scopus (479) Google Scholar, 35Graille M. Chaillet M. van Tilbeurgh H. J. Biol. Chem. 2008; 283: 7145-7154Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Lee H.H. Kim Y.S. Kim K.H. Heo I. Kim S.K. Kim O. Kim H.K. Yoon J.Y. Kim H.S. Kim do.J. Lee S.J. Yoon H.J. Kim S.J. Lee B.G. Song H.K. Kim V.N. Park C.M. Suh S.W. Mol. Cell. 2007; 27: 938-950Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). At present we have no experimental confirmation of the role of glucosylation of Hbs1 in Legionella intracellular biology or disease. However, it should be noted that both identified targets of Lgt1, namely eEF1A and Hbs1, represent important components of protein synthesis machinery in eukaryotic cells. In our previous studies we demonstrated that the glucosylation of eEF1A takes place following intoxication of EBL cells by Lgt1 (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). However we cannot exclude that modification of Hbs1 is also of importance for Lgt1-induced biological effects (e.g. inhibition of protein synthesis).The identification of a small peptide that is glucosylated by Lgt1 allowed us to study the stereochemical structure of the O-glycosidic bond formed. The anomeric configuration of the product of glucosylation can be either retained or inverted with respect to the donor co-substrate (37Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Annu. Rev. Biochem. 2008; 77: 521-555Crossref PubMed Scopus (1276) Google Scholar). Here we show by NMR spectroscopy that Lgt1 belongs to the retaining type of glucosyltransferases. Therefore, Lgt1 is similar to the clostridial glucosylating toxins, which glucosylate Rho and/or Ras GTPases in a retaining reaction (27Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 38Geyer M. Wilde C. Selzer J. Aktories K. Kalbitzer H.R. Biochemistry. 2003; 42: 11951-11959Crossref PubMed Scopus (32) Google Scholar, 39Vetter I.R. Hofmann F. Wohlgemuth S. Herrmann C. Just I. J. Mol. Biol. 2000; 301: 1091-1095Crossref PubMed Scopus (56) Google Scholar, 40Ziegler M.O. Jank T. Aktories K. Schulz G.E. J. Mol. Biol. 2008; 377: 1346-1356Crossref PubMed Scopus (49) Google Scholar). Sequence comparison of Lgt1 with clostridial toxins have shown some sequence similarity in the surrounding of the conserved DXD motif, which is involved in coordination of manganese ions and binding of the activated co-substrate UDP-glucose. It remains to be studied whether the structure of the catalytic domain of Lgt1 is related to clostridial glucosyltransferases. Elongation factor eEF1A, with two structurally highly similar isoforms eEF1A1 and eEF1A2, is a large GTP-binding protein, consisting of the N-terminal GTPase domain and two additional domains of β-barrel structure (26Andersen G.R. Nyborg J. Cold Spring Harb. Symp. Quant. Biol. 2001; 66: 425-437Crossref PubMed Scopus (15) Google Scholar). L. pneumophila glucosyltransferase Lgt1 glucosylates eEF1A at serine 53, which is located in the GTPase domain of the elongation factor (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). Here we studied the substrate recognition of eEF1A by Lgt1. Our studies show that domains 2 and 3 of eEF1A are fully dispensable for glucosylation by Lgt1. Moreover, efficient glucosylation by Lgt1 was observed with the decapeptide 50GKGSFKYAWV59 as a substrate. This suggests that the three-dimensional structure of eEF1A is not important for recognition by Lgt1. Many bacterial protein toxins are characterized by extremely high substrate specificity. These examples include C. difficile toxins A and B, which are at least distantly related to Lgt1 (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar), as well as other members of the glucosylating clostridial toxin family. They modify small GTPases of the Rho and/or Ras family in a highly specific manner, depending on the intact folding of the whole substrate protein (27Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). For example, change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin of C. sordellii. Similarly, substitution of phenylalanine 85 in RhoD with serine allowed its glucosylation by toxin B of C. difficile (28Jank T. Pack U. Giesemann T. Schmidt G. Aktories K. J. Biol. Chem. 2006; 281: 19527-19535Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In contrast, the cytotoxic necrotizing factors CNF of E. coli needs only a rather small Rho peptide of 20 residues to catalyze deamidation of the target glutamine residue (29Lerm M. Schmidt G. Goehring U.M. Schirmer J. Aktories K. J. Biol. Chem. 1999; 274: 28999-29004Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Even smaller is the minimum peptide of eEF1A, which is modified by Lgt1. It contains the sequence 50GKGSFKYAWV59 and forms the loop, which is a part of the helix-loop-helix structure of helices A* and A′ of eEF1A. Whereas glycine 50 is the first residue of this loop, the loop ends with lysine 55. Tyrosine 56 and the other residues of the peptide form the first turn of helix A′. Serine 53 is located in the middle of the loop and apparently is freely accessible at the surface of eEF1A. Even smaller truncations (e.g. KGSFKYAWV, GKGSFKYAW, and KGSFKYAW) were substrates for Lgt1, although glucosylation was less efficient. As one can deduce from the experiments, the COOH-terminal part of the peptide was more important for its substrate activity than the NH2-terminal part. In line with this notion were results of mutagenesis studies. Substitution of amino acid residues located COOH-terminally (e.g. lysine 55, tyrosine 56, alanine 57, or tryptophan 58) caused major reduction in glucosylation and substitution of phenylalanine 54 completely prevented glucosylation of the neighboring serine 53. Glucosylation of serine 53 of eEF1A inhibits protein synthesis (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). However, the functional consequences of the modification of serine 53 are not clear on the molecular level. Serine 53, which is glucosylated by Lgt1 in eEF1A, is not present in prokaryotic EF1A (known as EF-Tu) but conserved in many eukaryotic elongation factors 1A. Whereas glucosylation of the small GTPase RhoA by clostridial glucosyltransferases modifies threonine 37, which is essential for nucleotide binding and coordination of magnesium (30Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar), the glucosylation site (serine 53) in eEF1A is not directly involved in nucleotide binding. Glucosylation of RhoA and Ras by clostridial toxins inhibits the active conformation of the protein. The exposed location of serine 53 in eEF1A rather suggests that glucosylation of the elongation factor interferes with binding and/or interaction with other proteins or factors, eventually resulting in inhibition of protein synthesis. Interestingly, the rate of glucosylation of peptide 50GKGSFKYAWV59 was at least 20-fold higher than the glucosylation of a native full-length eEF1A or its recombinant G domain. This suggests that a specific conformation of eEF1A might be the preferred substrate of Lgt1. However, our attempts to find out conditions that cause a major increase in glucosylation by Lgt1 have been unsuccessful so far. The short peptide, which was recognized by Lgt1 as a substrate for glucosylation, prompted us to search for other proteins, harboring this sequence. A BLAST search identified Hsp70 subfamily B suppressor 1 (Hbs1) as a putative target for glucosylation. Recombinant Hbs1 was readily modified by Lgt1-induced glucosylation. Initially Hbs1 was identified in yeast screens as a 70-kDa heat shock cognate protein, which suppressed defects caused by a proteasome mutation in Saccharomyces cerevisiae (31Ohba M. FEBS Lett. 1994; 351: 263-266Crossref PubMed Scopus (25) Google Scholar). Later it was shown that Hbs1 possesses strong homology to release factor eRF3 (32Wallrapp C. Verrier S.B. Zhouravleva G. Philippe H. Philippe M. Gress T.M. Jean-Jean O. FEBS Lett. 1998; 440: 387-392Crossref PubMed Scopus (37) Google Scholar). Hbs1 and Hbs1-interacting eRF1-related factor Dom34 appear to be important for efficient protein synthesis in and growth of yeast cells under conditions of limiting translation initiation (33Carr-Schmid A. Pfund C. Craig E.A. Kinzy T.G. Mol. Cell. Biol. 2002; 22: 2564-2574Crossref PubMed Scopus (57) Google Scholar). In more recent reports, relationships between Hbs1 function and ribosomal mRNA translation were described (34Doma M.K. Parker R. Nature. 2006; 440: 561-564Crossref PubMed Scopus (479) Google Scholar, 35Graille M. Chaillet M. van Tilbeurgh H. J. Biol. Chem. 2008; 283: 7145-7154Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Lee H.H. Kim Y.S. Kim K.H. Heo I. Kim S.K. Kim O. Kim H.K. Yoon J.Y. Kim H.S. Kim do.J. Lee S.J. Yoon H.J. Kim S.J. Lee B.G. Song H.K. Kim V.N. Park C.M. Suh S.W. Mol. Cell. 2007; 27: 938-950Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). At present we have no experimental confirmation of the role of glucosylation of Hbs1 in Legionella intracellular biology or disease. However, it should be noted that both identified targets of Lgt1, namely eEF1A and Hbs1, represent important components of protein synthesis machinery in eukaryotic cells. In our previous studies we demonstrated that the glucosylation of eEF1A takes place following intoxication of EBL cells by Lgt1 (9Belyi Y. Niggeweg R. Opitz B. Vogelsgesang M. Hippenstiel S. Wilm M. Aktories K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16953-16958Crossref PubMed Scopus (110) Google Scholar). However we cannot exclude that modification of Hbs1 is also of importance for Lgt1-induced biological effects (e.g. inhibition of protein synthesis). The identification of a small peptide that is glucosylated by Lgt1 allowed us to study the stereochemical structure of the O-glycosidic bond formed. The anomeric configuration of the product of glucosylation can be either retained or inverted with respect to the donor co-substrate (37Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Annu. Rev. Biochem. 2008; 77: 521-555Crossref PubMed Scopus (1276) Google Scholar). Here we show by NMR spectroscopy that Lgt1 belongs to the retaining type of glucosyltransferases. Therefore, Lgt1 is similar to the clostridial glucosylating toxins, which glucosylate Rho and/or Ras GTPases in a retaining reaction (27Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 38Geyer M. Wilde C. Selzer J. Aktories K. Kalbitzer H.R. Biochemistry. 2003; 42: 11951-11959Crossref PubMed Scopus (32) Google Scholar, 39Vetter I.R. Hofmann F. Wohlgemuth S. Herrmann C. Just I. J. Mol. Biol. 2000; 301: 1091-1095Crossref PubMed Scopus (56) Google Scholar, 40Ziegler M.O. Jank T. Aktories K. Schulz G.E. J. Mol. Biol. 2008; 377: 1346-1356Crossref PubMed Scopus (49) Google Scholar). Sequence comparison of Lgt1 with clostridial toxins have shown some sequence similarity in the surrounding of the conserved DXD motif, which is involved in coordination of manganese ions and binding of the activated co-substrate UDP-glucose. It remains to be studied whether the structure of the catalytic domain of Lgt1 is related to clostridial glucosyltransferases. We thank Dr. C. R. Knudsen for eEF1A-expressing plasmids and helpful hints to establish the GTPase assay, Dr. G. R. Andersen for eEF1Bα-expressing plasmid and for providing protocol of eEF1A purification, and Dr. T. G. Kinzy for generous gift of the Hbs1 gene-containing plasmid. Supplementary Material Download .zip (.21 MB) Help with zip files Download .zip (.21 MB) Help with zip files" @default.
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• W2019410617 title "Region of Elongation Factor 1A1 Involved in Substrate Recognition by Legionella pneumophila Glucosyltransferase Lgt1" @default.
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