Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site.

West, Robert; Moss, Joel; Vaughan, Martha; Liu, T.; Liu, Teh‐Yung

doi:10.1016/s0021-9258(17)38585-x

Cited by 443 publications

(60 citation statements)

References 44 publications

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“…The traditional method to vesiculate cells to form GPMVs utilizes NEM that covalently modifies cysteine residues in proteins (29). While our data suggest that NEM treatment does not adversely impact β2AR functionality, we sought to address concerns for other receptors and Gα C-terminal peptides that might be affected by cysteine modifications [notably, the cysteine residue in Gαi that is the primary target of pertussis-toxin treatment (30)]. Hence, we used an alternate approach that triggers vesiculation using a bicine buffer that should not directly react with cellular proteins (31).…”

Section: Resultsmentioning

confidence: 99%

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Kim

Paulekas

Sadler

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Classical pharmacological models have incorporated an “intrinsic efficacy” parameter to capture system-independent effects of G protein–coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand “molecular efficacy” that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor–G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor–G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor–G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

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Section: Resultsmentioning

confidence: 99%

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Kim

Paulekas

Sadler

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…The requirement for NAD for action led to the discovery that diphtheria toxin catalyzes the NAD-dependent ADP-ribosylation of the elongation factor involved in eukaryotic protein synthesis (Honjo et al, 1968;Kandel et al, 1974). Other bacterial toxins, including cholera toxin and pertussis toxin, catalyze the ADP-ribosylations of the subunits of eukaryotic G proteins, leading to cell death (Moss and Vaughan, 1977;West et al, 1985). In these cases, the modification appears to be irreversible.…”

Section: Other Adp-ribosylationsmentioning

confidence: 99%

Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

Nordlund

Ludden

Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria

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The photosynthetic bacteria are remarkable for their range of metabolic capabilities and, as a result, the ability to survive under a wide range of environmental conditions, including nitrogen limitation. The photosynthetic bacteria are members of the alpha subdivision of the proteobacteria (Imhoff et al., 1984;Woese et al., 1984) and, with a single known exception, the wild-type strains of all purple sulfur bacteria and all purple non-sulfur bacteria are capable of N 2 fixation (Madigan et al., 1984). Most of the work that will be described in this chapter has been performed on a few species of purple, non-sulfur bacteria, including Rhodospirillum rubrum and Rhodobacter capsulatus. This chapter will focus on the biochemistry and physiology of N 2 fixation by photosynthetic bacteria and the regulation of nitrogenase activity by reversible ADP-ribosylation of the dinitrogenase reductase (Fe protein). ADP-ribosylation has also been found in Azospirillum and other genera and, where relevant, that work will be included. Other chapters in this series will cover the genes of the nif, anf and rnf regulons of the photosynthetic bacteria and the regulation of the expression of those genes. Although this chapter will discuss aspects of the nitrogenase enzyme, the mechanistic and structural details of nitrogenase will be covered in more detail in Volume 1 of this series.

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“…Moss et al [29] found that in the absence of Gi/oα a water molecule could serve as acceptor substrate and NAD-glycohydrolase activity could be readily measured by the release of [carbonyl- 14 C]nicotinamide from [carbonyl- 14 C]NAD with a K m for NAD of approximately 20 µM. However, the catalytic rate of G-protein ADP-ribosylation by PT is roughly ten times faster than that of NAD glycohydrolysis in the absence of G proteins [29], suggesting an active role of the acceptor substrate site, identified as the C-proximal cysteine residue located four amino acids from the carboxy-terminus [30], in catalysis.…”

Section: Mechanism Of Adp-ribosytransferase Activity Of Ptmentioning

confidence: 99%

The History of Pertussis Toxin

Locht

Antoine

2021

Toxins

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Besides the typical whooping cough syndrome, infection with Bordetella pertussis or immunization with whole-cell vaccines can result in a wide variety of physiological manifestations, including leukocytosis, hyper-insulinemia, and histamine sensitization, as well as protection against disease. Initially believed to be associated with different molecular entities, decades of research have provided the demonstration that these activities are all due to a single molecule today referred to as pertussis toxin. The three-dimensional structure and molecular mechanisms of pertussis toxin action, as well as its role in protective immunity have been uncovered in the last 50 years. In this article, we review the history of pertussis toxin, including the paradigm shift that occurred in the 1980s which established the pertussis toxin as a single molecule. We describe the role molecular biology played in the understanding of pertussis toxin action, its role as a molecular tool in cell biology and as a protective antigen in acellular pertussis vaccines and possibly new-generation vaccines, as well as potential therapeutical applications.

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Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site.

Cited by 443 publications

References 44 publications

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

The History of Pertussis Toxin

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