A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

Düvel, Juliane; Bertinetti, Daniela; Möller, Stefan; Schwede, Frank; Morr, Michael; Wissing, Josef; Radamm, Lena; Zimmermann, Bastian; Genieser, Hans‐Gottfried; Jänsch, Lothar; Häußler, Susanne

doi:10.1016/j.mimet.2011.11.015

Cited by 52 publications

(57 citation statements)

References 43 publications

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“…5). Ni-NTA agarose eluates from triplicate experiments for the isolation of the cross-linked complexes to the respective NosR-His 6ϫ , NorC-His 6ϫ , and NorB-His 6ϫ bait proteins were subsequently evaluated by LC-MS/MS as previously described (55). The eluted proteins with their attached copurified interaction partners were identified by quantitative mass spectrometry (AP-QMS) (56).…”

Section: Methodsmentioning

confidence: 99%

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Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

et al. 2016

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Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa. During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO 3؊ ¡ NO 2؊ ¡ NO ¡ N 2 O ¡ N 2 . Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment. IMPORTANCEThe processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.

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Section: Methodsmentioning

confidence: 99%

“…The following search parameters were used: enzyme, trypsin; maximum missed cleavages, 1; fixed modification, MMTS(C); variable modifications, oxidation (M); peptide tolerance, 10 ppm; and MS/MS tolerance, 0.4 Da. The results were evaluated and quantitated using Proteome Discoverer 1.4 (53,55,57).…”

Section: Methodsmentioning

confidence: 99%

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

et al. 2016

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“…STING binds c-di-GMP with a K D of 5 M and stimulates production of type I interferons by signaling through the TANK binding kinase 1 (TBK1) and the interferon regulatory transcription factor 3 (IRF3) (69). At least five groups have solved the crystal structure of STING (70,72,73,177,421) in complex with c-di-GMP (Fig. 5G).…”

Section: Cyclic Di-gmp As An Immunomodulatormentioning

confidence: 99%

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

Römling

Galperin

Gomelsky

2013

Microbiol Mol Biol Rev

1,500

1,698

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SUMMARY Twenty-five years have passed since the discovery of cyclic dimeric (3′→5′) GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger.

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“…Thus, identification of further classes of c-di-GMP binding domains can be expected, and many questions on the nature of the targets of c-di-GMP action are still open. We have recently applied a chemical proteomics approach employing a functionalized c-di-GMP analog to uncover novel candidate cdi-GMP binding proteins in the opportunistic bacterial pathogen Pseudomonas aeruginosa (37).…”

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Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs

et al. 2016

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High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein.

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A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

Cited by 52 publications

References 43 publications

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs

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