| 1. |
- Ernst, Stefan, et al.
(författare)
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Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination
- 2020
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Ingår i: Science Advances. - : American Association for the Advancement of Science. - 2375-2548. ; 6:20
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Tidskriftsartikel (refereegranskat)abstract
- The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)–choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.
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| 2. |
- Heller, Katharina, et al.
(författare)
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Covalent Protein Labeling by Enzymatic Phosphocholination
- 2015
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:35, s. 10327-10330
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Tidskriftsartikel (refereegranskat)abstract
- We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.
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| 3. |
- Heller, K, et al.
(författare)
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Enzymatic phosphocholination as a tool for protein labeling
- 2015
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 282, s. 12-12
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Posttranslational modification (PTM) of proteins is a versatile cellular process to regulate the activities of proteins. The high regioselectivity and catalysis rate of posttranslationally modifying enzymes utilizing high-energy precursors can potentially be exploited to equip proteins or peptide sequences with a label of choice site selectively and efficiently. We and others have recently described and analyzed a new reversible PTM called phosphocholination in which a phosphocholine group is transferred from a cytidine diphosphate choline (CDP-choline) to a serine residue of the small GTPase Rab1 [1–3]. The enzymes AnkX and Lem3 catalyze the modification and the corresponding demodification reactions, respectively. Interestingly, we could demonstrate that the modifying enzyme AnkX only requires a short amino acid sequence for substrate recognition. Therefore, we envision AnkX as a tool for the site directed labeling of target proteins. Here we report on the progress of developing a novel reversible protein labeling strategy based on the enzymes AnkX and Lem3 and on derivatives of CDP-choline. We demonstrate the optimization of AnkX and Lem3 enzyme activities and the identification of optimal and minimal peptide target sequences. Results indicate that indeed arbitrary proteins of interest can be functionalized with phosphocholine derivatives. In summary, this work yields first insights into the development of a CDP-choline based fully reversible protein labeling strategy.
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| 4. |
- Kaspers, Marietta S., et al.
(författare)
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Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.
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| 5. |
- Ochtrop, Philipp, et al.
(författare)
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Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization
- 2019
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Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 20:18, s. 2336-2340
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Tidskriftsartikel (refereegranskat)abstract
- Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5x10(2) to 1.8x10(4) m(-1) s(-1). Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling.
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| 6. |
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| 7. |
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| 8. |
- Ochtrop, Philipp, 1986-
(författare)
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Selective protein functionalisation via enzymatic phosphocholination
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes ultimately determining cellular function. Therefore, to develop a complete understanding of cellular processes, obtaining knowledge about protein function and interaction at a molecular level is critical. Consequently, the investigation of proteins in their native environment or in partially purified mixtures is a major endeavour in modern life sciences. Due to their high chemical similarity, the inherent problem of studying proteins in complex mixtures is to specifically differentiate one protein of interest from the bulk of other proteins. Site-specific protein functionalisation strategies have become an indispensable tool in biochemical- and cell biology studies. This thesis presents the development of a new enzymatic site-specific protein functionalisation strategy that is based on the reversible covalent phosphocholination of short amino acid sequences in intact proteins. A synthetic strategy has been established that allows access to functionalised CDP-choline derivatives carrying fluorescent reporter groups, affinity tags or bioorthogonal handles. These CDP-choline derivatives serve as co-substrates for the bacterial phosphocholinating enzyme AnkX from Legionella pneumophila, which transfers a phosphocholine moiety to the switch II region of its native target protein Rab1b during infection. We identified the octapeptide sequence TITSSYYR as the minimum recognition sequence required to direct the AnkX catalysed phosphocholination and demonstrated the functionalisation of proteins of interest carrying this recognition tag at the N- or C-terminus as well as in internal loop regions. Moreover, this covalent modification can be hydrolytically reversed by the action of the Legionella enzyme Lem3, which makes the labeling strategy the first example of a covalent and reversible approach that is fully orthogonal to current existing methodologies. Thus, the here presented protein functionalisation approach holds the potential to increase the scope of possible labeling strategies in complex biological systems. In addition to the labeling of tagged target proteins, a CDP-choline derivative equipped with a biotin affinity-tag was synthesised and used in pull-down experiments to investigate the substrate scope of AnkX and to elucidate the role of protein phosphocholination during Legionella pneumophila infection.
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| 9. |
- Paulsen, Marianne H., et al.
(författare)
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An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2-amino acids with activity against multiresistant bacteria
- 2018
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Ingår i: Journal of Peptide Science. - : John Wiley & Sons. - 1075-2617 .- 1099-1387. ; 24:10
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Tidskriftsartikel (refereegranskat)abstract
- The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys‐β2,2‐Xaa‐Lys) containing one lipophilic β2,2-amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum β-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8μg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic β2,2-amino acids form a valuable scaffold for designing novel antimicrobial agents.
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