Molecular basis for the evolved instability of a human G-protein coupled receptor

Chamness, Laura M.; Zelt, Nathan B.; Harrington, Haley R.; Kuntz, Charles P.; Bender, Brian J.; Penn, Wesley D.; Ziarek, Joshua J.; Meiler, Jens; Schlebach, Jonathan P.

doi:10.1016/j.celrep.2021.110046

Cited by 11 publications

(35 citation statements)

References 61 publications

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“…The helix 8 and C-tail of GPCRs are comparaMvely lesser studied receptor regions despite their importance in cell surface trafficking, acMvaMon, and transducer interacMons. [5][6][7][8][9][10][11][12][13][14][15][16] Our study explores the structural and dynamic properMes of the isolated rat NTS1 helix 8 and C-tail in soluMon. We demonstrated that a hydrophobic membrane-like environment is required to stabilize helicity within the helix 8 region of the GPCR, while the C-tail remains generally unstructured.…”

Section: Discussionmentioning

confidence: 99%

Insights on the GPCR helix 8 solution structure and orientation using a neurotensin receptor 1 peptide

Bower,

Robson,

Ziarek

2024

Preprint

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G-protein coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminus. Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the C-tail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and C-terminus (H8-Ctail) to investigate its structural stability, conformational dynamics and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix – even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement (PRE) NMR. Taken together, our studies reveal the H8C-tail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

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Section: Discussionmentioning

confidence: 99%

Insights on the GPCR helix 8 solution structure and orientation using a neurotensin receptor 1 peptide

Bower,

Robson,

Ziarek

2024

Preprint

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“…Plasmids were purified using a ZymoPURE Midiprep Kit (Zymo Research, Irvine, CA). DMS experiments were carried out using a previously described pcDNA5 FRT vector containing mGnRHR cDNA bearing an N-terminal influenza hemagglutinin (HA) epitope, followed by an internal ribosome entry site (IRES) and eGFP sequence, which was further modified to be compatible with recombination-based DMS approaches (Chamness et al, 2021). First, an attB recombination site was inserted in place of the CMV promoter by InFusion HD Cloning (Takara Bio, Shiga, Japan).…”

Section: Discussionmentioning

confidence: 99%

“…To survey the epistatic interactions formed by these mutations, we generated a series of genetic libraries consisting of 1,615 missense variants in the background of V276T, W107A, and WT mGnRHR. To ensure adequate dynamic range in the downstream assay, we created these variants in the cDNA of mGnRHR, which exhibits intermediate, tunable expression (Chamness et al, 2021). Briefly, we used a structural homology model of mGnRHR to select 85 residues distributed across the loops and helices of mGnRHR.…”

Section: Mutational Library Designmentioning

confidence: 99%

“…Expression Measurements of Individual GnRHR Variants HEK283T cells were obtained from ATCC and grown under the same conditions described above. GnRHR variants were transiently expressed in these cells, and the plasma membrane and intracellular expression of these variants was measured by flow cytometry, as previously described (Chamness et al, 2021). The reported expression levels represent measurements from three biological replicates.…”

Section: Plasma Membrane Expression and Epistasis Measurementsmentioning

confidence: 99%

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Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants

Chamness,

Kuntz,

McKee

et al. 2023

Preprint

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Many eukaryotic membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for mammalian gonadotropin-releasing hormone receptors (GnRHRs), which are G protein-coupled receptors involved in reproductive steroidogenesis. We recently demonstrated that evolutionary modifications within mammalian GnRHRs appear to have coincided with adaptive changes in cotranslational folding efficiency. Though changes in protein stability are known to shape evolutionary interactions, it is unclear how the energetic drivers of cotranslational folding in the membrane may modify epistatic interactions. We therefore surveyed the pairwise epistatic interactions that modify the expression of two destabilized GnRHR variants bearing mutations that selectively compromise either its membrane topology (V276T) or its native tertiary structure (W107A). Using deep mutational scanning (DMS), we evaluated how the effects of these mutations on the expression of the mature form of the protein at the plasma membrane are modified by hundreds of secondary mutations. A focused analysis of 251 mutants with high-quality measurements in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the degree to which they destabilize the protein and the mechanism of their destabilization. An unsupervised learning analysis shows that V276T forms predominantly negative epistatic interactions that are most pronounced among destabilizing mutations within soluble loop regions. In contrast, W107A forms interactions with mutations in both loops and transmembrane domains that skew positive as a result of the diminishing impact of the destabilizing mutations in the context of an already unstable variant. These findings provide general insights into how pairwise epistasis is remodeled by conformational defects in membrane proteins and, more generally, in unstable proteins.

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“…Notably, the N-terminal, already inserted parts of multispanning proteins may interact with EMC cotranslationally, potentially facilitating a swift handover(Shurtleff et al, 2018;Wu et al, 2023).The physiological importance of this insertion mechanism, which likely serves hundreds of MPs in every proteome, is underscored by its implications in disease. Misinsertion of MPs, leading to misfolding and loss of function(Chamness et al, 2021;Marshall et al, 2016), has been implicated in various genetic diseases(Coelho et al, 2019;Roushar et al, 2019;Sun &…”

mentioning

confidence: 99%

Membrane protein sequence features direct post-translational insertion

Kalinin,

Peled-Zehavi,

Barshap

et al. 2023

Preprint

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The proper folding of multispanning membrane proteins (MPs) hinges on the accurate insertion of their transmembrane helices (TMs) into the membrane. Predominantly, TMs are inserted during protein translation, via a conserved mechanism centered around the Sec translocon. Our study reveals that the C-terminal TMs (cTMs) of numerous MPs across various organisms bypass this cotranslational route, necessitating an alternative posttranslational insertion strategy. We demonstrate that evolution has refined the hydrophilicity and length of these proteins’ C-terminal tails to optimize cTM insertion. Alterations in the C-tail sequence disrupt cTM insertion in bothE. coliand human, leading to protein defects, loss of function, and genetic diseases. InE. coli, we identify YidC, a member of the widespread Oxa1 family, as the insertase facilitating cTMs insertion, with C-tail mutations disrupting the productive interaction of cTMs with YidC. Thus, MP sequences are fine-tuned for effective collaboration with the cellular biogenesis machinery, ensuring proper membrane protein folding.

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Molecular basis for the evolved instability of a human G-protein coupled receptor

Cited by 11 publications

References 61 publications

Insights on the GPCR helix 8 solution structure and orientation using a neurotensin receptor 1 peptide

Insights on the GPCR helix 8 solution structure and orientation using a neurotensin receptor 1 peptide

Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants

Membrane protein sequence features direct post-translational insertion

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