Lysine acylations, a family of diverse protein modifications varying in acyl‐group length, charge, and saturation, are linked to many important physiological processes. Only a small set of ...substrate‐promiscuous lysine acetyltransferases and deacetylases (KDACs) install and remove this vast variety of modifications. Engineered KDACs that remove only one type of acylation would help to dissect the different contributions of distinct acylations. We developed a bacterial selection system for the directed evolution of KDACs and identified variants up to 400 times more selective for butyryl‐lysine compared to crotonyl‐lysine. Structural analyses revealed that the enzyme adopts different conformational states depending on the type of acylation of the bound peptide. We used the butyryl‐selective KDAC variant to shift the cellular acylation spectrum towards increased lysine crotonylation. These new enzymes will help in dissecting the roles of different lysine acylations in cell physiology.
Ch‐ch‐changes: Cellular proteins exhibit complex lysine acylation patterns, which are reversed by a small set of substrate‐promiscuous lysine deacetylases (KDACs). A strategy was developed to create KDAC variants with increased selectivity towards acetyl (Ac) over butyryl (Bu) and crotonyl (Cr) modifications through directed evolution by using a novel selection system. These engineered KDACs can be used to dissect the roles of different lysine acylations in cells.
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•Unnatural amino acids provide a plethora of novel functionalities to study protein function.•Crosslinker UAAs are being used to monitor protein–protein interactions in living ...cells.•Genetically encoded post-translational modifications enable functional studies.•Bioorthogonal handles are being used to label and engineer proteins.
The expansion of the genetic code for the incorporation of unnatural amino acids (UAAs) in proteins of bacteria, yeasts, mammalian cells or whole animals provides molecular and structural biologists with an amazing kit of novel tools. UAAs can be used to investigate the structure and dynamics of proteins, to study their interactions or to control their activity in living cells. Incorporation of UAAs with bioorthogonal reactivity facilitates the site-specific installation of labels for spectroscopy and microscopy. Light-activatable crosslinker UAAs can be used to trap interacting molecules in living cells with a precision almost at the structural level. Post-translational modifications such as lysine acetylation and serine phosphorylation can be directly encoded to analyse their impact on protein function, and caging groups can be installed on critical residues to create light-activatable proteins. In this review we highlight recent applications of this technology to investigate protein function.
Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function, such as the activity, localization, and stability of enzymes. Mass spectrometric ...identification of lysine acylations has witnessed tremendous improvements in sensitivity over the last decade, facilitating the discovery of thousands of lysine acylation sites in proteins involved in all essential cellular functions across organisms of all domains of life. However, the vast majority of currently known acylation sites are of unknown function. Semi‐synthetic methods for installing lysine derivatives are ideally suited for in vitro experiments, while genetic code expansion (GCE) allows the installation and study of such lysine modifications, especially their dynamic properties, in vivo. An overview of the current state of the art is provided, and its potential is illustrated with case studies from recent literature. These include the application of engineered enzymes and GCE to install lysine modifications or photoactivatable crosslinker amino acids. Their use in the context of central metabolism, bacterial and viral pathogenicity, the cytoskeleton and chromatin dynamics, is investigated.
Genetic code expansion facilitates the production of proteins containing defined lysine acylations. These proteins can then be used to study the impact of the modification on their properties in vitro or on the physiology of the cell. This review summarizes recent technological developments and provides illustrative examples of their application.
Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs) by the multiple peptide resistance factor (MprF) protein. MprF plays a crucial role in ...Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with l-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed trans-membrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG) production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help to employ MprF as a target for new anti-virulence drugs.
Abstract
TRIM21 (‘tripartite motif containing protein 21’, Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 ...mediates intracellular neutralisation of antibody coated viruses, a process called ADIN (antibody dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity.
Characterisation of a cellular self defence mechanism where antibodies attached to intracellular Salomonellae are recognised by the cell and subsequently triggers the clearence of the intruder.
Characterisation of a cellular self defence mechanism where antibodies attached to intracellular Salomonellae are recognised by the cell and subsequently triggers the clearence of the intruder.
Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear ...membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions
in vivo
. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages.
Lysinacylierungen treten in vielen wichtigen physiologischen Prozessen auf. Nur eine kleine Anzahl Substrat‐promisker Lysin‐Acetyltransferasen und ‐Deacetylasen (KDACs) installiert und entfernt diese ...zahlreichen Modifikationen. Mit evolvierten KDACs, die nur bestimmte Acylierungen entfernen können, könnte die physiologische Bedeutung verschiedener Acylierungen für die Zelle analysiert werden. Wir haben ein bakterielles Selektionssystem für die gerichtete Evolution von KDACs entwickelt, mit dessen Hilfe wir Varianten identifizieren konnten, die Butyrylgruppen bis zu 400‐mal effizienter von Lysin entfernen als Crotonylreste. Strukturanalysen ergaben, dass das Enzym, abhängig von der Acylierung des Substrats, einen aktive oder inaktive Konformation annimmt. Mithilfe einer Butyryl‐selektiven KDAC‐Variante war es möglich, das zelluläre Acylierungsspektrum in Richtung erhöhter Lysincrotonylierung zu verschieben. Unser Selektionssystem ermöglicht es, KDAC‐Enzyme zu evolvieren, um die physiologische Signifikanz einzelner Acyllysinmodifikationen auf die Zelle zu untersuchen.
Zelluläre Proteine zeigen komplexe Lysinacylierungsmuster, die von einem kleinen Satz Substrat‐promisker Lysindeacetylasen (KDACs) reguliert werden. Mit einer neuen Strategie lassen sich KDAC‐Varianten mit erhöhter Selektivität gegenüber spezifischen Acylierungen durch gerichtete Evolution unter Verwendung eines neuartigen Selektionssystems erhalten. Diese künstlichen KDACs eignen sich zur Analyse der Rollen verschiedener Lysinacylierungen in Zellen.
•Unnatural amino acids (UAAs) offer unique avenues to study protein structure.•UAAs are ideal to study chromatin dynamics and epigenetic mechanisms.•UAAs help to unravel protein–protein interactions ...in cells by photo-crosslinking.•UAAs facilitate the biosynthesis of proteins with posttranslational modifications.
The genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technology provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein–protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells.
The installation of unnatural amino acids into proteins of living cells is an enabling technology that facilitates an enormous number of applications. UV-activatable crosslinker amino acids allow the ...formation of a covalent bond between interaction partners in living cells with nearly perfect spatial and temporal control. Here, we describe how this method can be employed to map chromatin interactions and to follow these interactions across the cell cycle in synchronized yeast populations. This method thereby provides unprecedented insights into the molecular events controlling chromatin reorganization in mitosis. As similar tools are available for other organisms, it should be possible to derive similar strategies for these and for other synchronizable processes.
Synthetic biology is the attempt to apply the concepts of engineering to biological systems with the aim to create organisms with new emergent properties. These organisms might have desirable novel ...biosynthetic capabilities, act as biosensors or help us to understand the intricacies of living systems. This approach has the potential to assist the discovery and production of pharmaceutical compounds at various stages. New sources of bioactive compounds can be created in the form of genetically encoded small molecule libraries. The recombination of individual parts has been employed to design proteins that act as biosensors, which could be used to identify and quantify molecules of interest. New biosynthetic pathways may be designed by stitching together enzymes with desired activities, and genetic code expansion can be used to introduce new functionalities into peptides and proteins to increase their chemical scope and biological stability. This review aims to give an insight into recently developed individual components and modules that might serve as parts in a synthetic biology approach to pharmaceutical biotechnology.
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