Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP

Bacterial protein glycosylation affects numerous cellular properties, including physiology and pathogenicity. The transfer of carbohydrates to a nitrogen atom is known as N glycosylation and almost exclusively occurs on asparagine side chains. In contrast, EarP represents a novel type of arginine-modifying glycosyltransferases. This enzyme uses TDP β-L-rhamnose as a donor substrate to activate the specialized translation elongation factor P (EF-P) in about 10 % of sequenced bacteria, including the clinically relevant species Pseudomonas aeruginosa and Neisseria meningitidis. The post-translational modification of EF-P is crucial for bacterial fitness and also constitutes a prerequisite for virulence. As the amido group of asparagine and the arginine guanidinium are chemically distinct, the activation of the latter might be based on a so far unsolved molecular mechanism. Consequently, the structural characterization of EarP and its products is of clinical and functional importance. In this regard, NMR analyses unambiguously identified the product of the glycosylation reaction as α-rhamnosyl-arginine. Thus, EarP inverts the anomeric configuration of rhamnose during the reaction. Anomer-specific mono-rhamnosyl-arginine-containing peptides were synthetized and used to raise antibodies against the modified side chain. These immunoglobulins were characterized with respect to their sensitivity and specificity towards the target epitope and used to determine enzyme kinetics of EarP. X-ray crystallography identified EarP as a member of the inverting GT-B superfamily and revealed the site for donor binding. Bioinformatic and mutant analyses elucidated the functional significance of several amino acids in orienting the nucleotide sugar and demonstrated the importance of two highly conserved aspartates for catalysis. Additionally, NMR titration experiments revealed that EarP mainly binds the N-terminal β barrel domain of its acceptor substrate EF-P. This information was utilized to generate the first synthetic target for EarP-mediated protein modification. The structurally but not sequentially related EF-P homologue from E. coli is naturally activated by lysylation of a lysine side chain. Successive mutation not only allowed modification but also activation of E. coli EF-P by the non-cognate and EarP-mediated rhamnosylation. This thesis provides new insights into the structure-function relationship of inverting arginine glycosylation. Additionally, it lays the groundwork for the application of EarP in synthetic biology and clinical research.Die Glykosylierung bakterieller Proteine beeinflusst zahlreiche zelluläre Eigenschaften wie Physiologie und Pathogenität. Die Übertragung von Kohlenhydraten auf ein Stickstoffatom wird als N-Glykosylierung bezeichnet und erfolgt fast ausschließlich an Asparagin-Seitenketten. Im Gegensatz dazu gehört EarP zu einer neuen Klasse von Arginin-modifizierenden Glykosyltransferasen. In etwa 10 % der sequenzierten Bakterien, einschließlich der klinisch relevanten Spezies Pseudomonas aeruginosa und Neisseria meningitidis, verwendet dieses Enzym TDP-β-L-rhamnose als Donorsubstrat zur Aktivierung des spezialisierten Translationselongationsfaktors P (EF-P). Die post-translationale Modifikation von EF-P ist von entscheidender Bedeutung für die bakterielle Fitness und eine Voraussetzung für Virulenz. Da die Amidogruppe von Asparagin und die Guanidinogruppe von Arginin chemisch unterschiedlich sind, erfolgt die Aktivierung der letzteren durch einen bisher unerforschten molekularen Mechanismus. Folglich ist die strukturelle Charakterisierung von EarP und seinen Katalyseprodukten sowohl von medizinischer als auch funktioneller Bedeutung. Mittels NMR wurde zunächst das Produkt der Glykosylierungsreaktion von EarP eindeutig als α-Rhamnosyl-Arginin identifiziert. Somit invertiert EarP die anomere Konfiguration von Rhamnose während der Reaktion. Anomer-spezifische mono-Rhamnosyl-Arginin enthaltende Peptide wurden synthetisiert und zur Generierung von Antikörpern verwendet. Diese Immunglobuline wurden hinsichtlich Sensitivität und Spezifität gegenüber dem Epitop charakterisiert und zur Bestimmung der Enzymkinetik von EarP verwendet. Die Kristallstrukturanalyse von EarP ermöglichte nicht nur eine Zuordnung des Enzyms zur Superfamilie der invertierenden GT-B-Glykosyltransferasen, sondern zeigte auch die Position der Donorbindestelle auf. Weitere bioinformatische und Mutagenese-basierte Studien führten zur Identifizierung von zwei für die Katalyse wichtigen Aspartaten sowie von mehreren Aminosäuren, die für die Orientierung des Nukleotidzuckers von Bedeutung sind. NMR-Titrationen ergaben, dass EarP hauptsächlich die N-terminale β-Barreldomäne des Akzeptorsubstrates EF-P bindet. Diese Information wurde verwendet, um den ersten synthetischen Akzeptor für eine EarP-vermittelte Proteinmodifikation zu generieren. Das strukturell, aber nicht sequentiell verwandte EF-P-Homolog von E. coli wird natürlicherweise durch Lysylierung einer Lysin-Seitenkette aktiviert. Infolge sukzessiver Aminsoäureaustausche wurde nicht nur die Modifikation von E. coli EF-P durch eine EarP vermittelte Rhamnosylierung erreicht, sondern auch die Aktivierung dieses Elongationsfaktors. Diese Arbeit liefert somit neue Erkenntnisse über die Struktur-Funktionsbeziehung der invertierenden Arginin-Glykosylierung. Darüber hinaus legt sie den Grundstein für die Anwendung von EarP in der Synthetischen Biologie und der klinischen Forschung

Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP

Bacterial protein glycosylation affects numerous cellular properties, including physiology and pathogenicity. The transfer of carbohydrates to a nitrogen atom is known as N glycosylation and almost exclusively occurs on asparagine side chains. In contrast, EarP represents a novel type of arginine-modifying glycosyltransferases. This enzyme uses TDP β-L-rhamnose as a donor substrate to activate the specialized translation elongation factor P (EF-P) in about 10 % of sequenced bacteria, including the clinically relevant species Pseudomonas aeruginosa and Neisseria meningitidis. The post-translational modification of EF-P is crucial for bacterial fitness and also constitutes a prerequisite for virulence. As the amido group of asparagine and the arginine guanidinium are chemically distinct, the activation of the latter might be based on a so far unsolved molecular mechanism. Consequently, the structural characterization of EarP and its products is of clinical and functional importance. In this regard, NMR analyses unambiguously identified the product of the glycosylation reaction as α-rhamnosyl-arginine. Thus, EarP inverts the anomeric configuration of rhamnose during the reaction. Anomer-specific mono-rhamnosyl-arginine-containing peptides were synthetized and used to raise antibodies against the modified side chain. These immunoglobulins were characterized with respect to their sensitivity and specificity towards the target epitope and used to determine enzyme kinetics of EarP. X-ray crystallography identified EarP as a member of the inverting GT-B superfamily and revealed the site for donor binding. Bioinformatic and mutant analyses elucidated the functional significance of several amino acids in orienting the nucleotide sugar and demonstrated the importance of two highly conserved aspartates for catalysis. Additionally, NMR titration experiments revealed that EarP mainly binds the N-terminal β barrel domain of its acceptor substrate EF-P. This information was utilized to generate the first synthetic target for EarP-mediated protein modification. The structurally but not sequentially related EF-P homologue from E. coli is naturally activated by lysylation of a lysine side chain. Successive mutation not only allowed modification but also activation of E. coli EF-P by the non-cognate and EarP-mediated rhamnosylation. This thesis provides new insights into the structure-function relationship of inverting arginine glycosylation. Additionally, it lays the groundwork for the application of EarP in synthetic biology and clinical research.Die Glykosylierung bakterieller Proteine beeinflusst zahlreiche zelluläre Eigenschaften wie Physiologie und Pathogenität. Die Übertragung von Kohlenhydraten auf ein Stickstoffatom wird als N-Glykosylierung bezeichnet und erfolgt fast ausschließlich an Asparagin-Seitenketten. Im Gegensatz dazu gehört EarP zu einer neuen Klasse von Arginin-modifizierenden Glykosyltransferasen. In etwa 10 % der sequenzierten Bakterien, einschließlich der klinisch relevanten Spezies Pseudomonas aeruginosa und Neisseria meningitidis, verwendet dieses Enzym TDP-β-L-rhamnose als Donorsubstrat zur Aktivierung des spezialisierten Translationselongationsfaktors P (EF-P). Die post-translationale Modifikation von EF-P ist von entscheidender Bedeutung für die bakterielle Fitness und eine Voraussetzung für Virulenz. Da die Amidogruppe von Asparagin und die Guanidinogruppe von Arginin chemisch unterschiedlich sind, erfolgt die Aktivierung der letzteren durch einen bisher unerforschten molekularen Mechanismus. Folglich ist die strukturelle Charakterisierung von EarP und seinen Katalyseprodukten sowohl von medizinischer als auch funktioneller Bedeutung. Mittels NMR wurde zunächst das Produkt der Glykosylierungsreaktion von EarP eindeutig als α-Rhamnosyl-Arginin identifiziert. Somit invertiert EarP die anomere Konfiguration von Rhamnose während der Reaktion. Anomer-spezifische mono-Rhamnosyl-Arginin enthaltende Peptide wurden synthetisiert und zur Generierung von Antikörpern verwendet. Diese Immunglobuline wurden hinsichtlich Sensitivität und Spezifität gegenüber dem Epitop charakterisiert und zur Bestimmung der Enzymkinetik von EarP verwendet. Die Kristallstrukturanalyse von EarP ermöglichte nicht nur eine Zuordnung des Enzyms zur Superfamilie der invertierenden GT-B-Glykosyltransferasen, sondern zeigte auch die Position der Donorbindestelle auf. Weitere bioinformatische und Mutagenese-basierte Studien führten zur Identifizierung von zwei für die Katalyse wichtigen Aspartaten sowie von mehreren Aminosäuren, die für die Orientierung des Nukleotidzuckers von Bedeutung sind. NMR-Titrationen ergaben, dass EarP hauptsächlich die N-terminale β-Barreldomäne des Akzeptorsubstrates EF-P bindet. Diese Information wurde verwendet, um den ersten synthetischen Akzeptor für eine EarP-vermittelte Proteinmodifikation zu generieren. Das strukturell, aber nicht sequentiell verwandte EF-P-Homolog von E. coli wird natürlicherweise durch Lysylierung einer Lysin-Seitenkette aktiviert. Infolge sukzessiver Aminsoäureaustausche wurde nicht nur die Modifikation von E. coli EF-P durch eine EarP vermittelte Rhamnosylierung erreicht, sondern auch die Aktivierung dieses Elongationsfaktors. Diese Arbeit liefert somit neue Erkenntnisse über die Struktur-Funktionsbeziehung der invertierenden Arginin-Glykosylierung. Darüber hinaus legt sie den Grundstein für die Anwendung von EarP in der Synthetischen Biologie und der klinischen Forschung

Synthetic post-translational modifications of elongation factor P using the ligase EpmA

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-beta-lysine to the epsilon-amino group of lysine 34 of the translation elongation factor P (EF-P) inEscherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants onin vitrotranslation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-beta-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA

Comparison of the functional properties of trimeric and monomeric CaiT of Escherichia coli

Secondary transporters exist as monomers, dimers or higher state oligomers. The significance of the oligomeric state is only partially understood. Here, the significance of the trimeric state of the L-carnitine/gamma-butyrobetaine antiporter CaiT of Escherichia coli was investigated. Amino acids important for trimer stability were identified and experimentally verified. Among others, CaiT-D288A and -D288R proved to be mostly monomeric in detergent solution and after reconstitution into proteoliposomes, as shown by blue native gel electrophoresis, gel filtration, and determination of intermolecular distances. CaiT-D288A was fully functional with kinetic parameters similar to the trimeric wild-type. Significant differences in amount and stability in the cell membrane between monomeric and trimeric CaiT were not observed. Contrary to trimeric CaiT, addition of substrate had no or only a minor effect on the tryptophan fluorescence of monomeric CaiT. The results suggest that physical contacts between protomers are important for the substrate-induced changes in protein fluorescence and the underlying conformational alterations

Switching the Post-translational Modification of Translation Elongation Factor EF-P

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands β3/β4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli. Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF- Ps from these two model organisms we demonstrate the importance of the β3/β4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-L-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design

Switching the Post-translational Modification of Translation Elongation Factor EF-P

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands β3/β4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli. Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF-Ps from these two model organisms we demonstrate the importance of the β3/β4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation

Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first ArgRha specific antibody

A previously discovered posttranslational modification strategy – arginine rhamnosylation – is essential for elongation factor P (EF-P) dependent rescue of polyproline stalled ribosomes in clinically relevant species such as Pseudomonas aeruginosa and Neisseria meningitidis. However, almost nothing is known about this new type of N-linked glycosylation. In the present study we used NMR spectroscopy to show for the first time that the α anomer of rhamnose is attached to Arg32 of EF-P, demonstrating that the corresponding glycosyltransferase EarP inverts the sugar of its cognate substrate dTDP-β-L-rhamnose. Based on this finding we describe the synthesis of an α-rhamnosylated arginine containing peptide antigen in order to raise the first anti-rhamnosyl arginine specific antibody (anti-ArgRha). Using ELISA and Western Blot analyses we demonstrated both its high affinity and specificity without any cross-reactivity to other N-glycosylated proteins. Having the anti-ArgRha at hand we were able to visualize endogenously produced rhamnosylated EF-P. Thus, we expect the antibody to be not only important to monitor EF-P rhamnosylation in diverse bacteria but also to identify further rhamnosyl arginine containing proteins. As EF-P rhamnosylation is essential for pathogenicity, our antibody might also be a powerful tool in drug discovery

Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design