Protons as Second Messenger Regulators of G Protein Signaling

Isom, Daniel G.; Sridharan, Vishwajith; Baker, Rachael; Clement, Sarah T.; Smalley, David M.; Dohlman, Henrik G.

doi:10.1016/j.molcel.2013.07.012

Cited by 77 publications

(136 citation statements)

References 44 publications

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“…3B) coincides with the loss of an internal proton-sensing network located at the interface of the Ras and helical domains of Gαs (Fig. 3B) (22). Likewise, the network buried within M2R is linked to a network of ionizable residues residing in its bound G protein-mimetic nanobody (Fig.…”

Section: Significancementioning

confidence: 60%

“…functions, including oxygen delivery by hemoglobin (13), pH-gated ion transport (19,20), and signaling by pH-sensing (protonactivated) GPCRs (21) and G proteins (22).…”

Section: Significancementioning

confidence: 99%

“…However, the relationship between ionizable groups and protein function can be studied computationally using physics-based models of protein electrostatics (23). To accelerate these efforts, we have developed an analytics-based approach, known as "pHinder" (22), for identifying functionally important ionizable interactions in proteins. pHinder differs from other structure-based electrostatics calculations by considering only the spatial organization (i.e., topology) of ionizable groups in a protein.…”

Section: Significancementioning

confidence: 99%

“…Previously we have used pHinder to identify networks of electrostatic interactions buried in G protein α (Gα) subunits, the principal transducers of GPCR signals (22). Upon GPCR activation, the Gα subunit undergoes substantial structural changes as it exchanges GTP for GDP.…”

Section: Significancementioning

confidence: 99%

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Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Isom¹,

Dohlman

2015

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

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Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light-and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligandbinding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.7-transmembrane receptor | G protein-coupled receptor | buried charge | structural bioinformatics | molecular evolution S even-transmembrane receptors (7TMRs) are present in all domains of life. In archaea and bacteria these receptors convert light energy into transmembrane ion gradients or intracellular signaling cascades. In humans 7TMRs comprise a family of more than 800 G protein-coupled receptors (GPCRs) that detect extracellular signals such as odorants, taste, light, hormones, and neurotransmitters. Although microbial and eukaryotic 7TMRs lack sequence similarity, their 3D folds share a degree of similarity that often is observed in remote evolutionary relationships identified by structure comparison algorithms (1).Recent breakthroughs in membrane protein crystallography have advanced our understanding of GPCR function by providing models of unactivated and activated receptor conformations (2-8). However, structure-based approaches for systematically quantifying differences between GPCR activation states are lacking. Here we introduce a computational approach that correlates GPCR activation with networks of electrostatic interactions in the receptor core. We show that membrane-spanning networks of ionizable residues, which we find are a common feature of microbial 7TMRs, also represent a unique signature of GPCR activation that is likely to be essential to receptor function.The molecular changes that accompany 7TMR activation were studied initially in bacteriorhodopsin and rhodopsin, the first microbial 7TMR and GPCR to be crystallized (9-11). More recently, a rapid expansion of GPCR structural information has revealed several molecular switches and structural features that are thought to be indicative of receptor activation. These include the ionic loc...

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Section: Significancementioning

confidence: 60%

“…functions, including oxygen delivery by hemoglobin (13), pH-gated ion transport (19,20), and signaling by pH-sensing (protonactivated) GPCRs (21) and G proteins (22).…”

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Isom¹,

Dohlman

2015

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

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“…Consequently, any pH change can cause a shift of the equilibrium, enriching protonated/relaxed species or deprotonated/retracted species. pHsensitive conformational switches have been reported for many other proteins (25)(26)(27). It has also been reported that K48-linked diubiquitin switches from a closed conformation to a predominant open conformation at pH 4.5 (28,29).…”

Section: Discussionmentioning

confidence: 83%

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Gong

et al. 2017

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

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Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

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