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Thermodynamic architecture and conformational plasticity of GPCRs.

ABSTRACT: G-protein-coupled receptors (GPCRs) are ubiquitous integral membrane proteins involved in diverse cellular signaling processes. Here, we carry out a large-scale ensemble thermodynamic study of 45 ligand-free GPCRs employing a structure-based statistical mechanical framework. We find that multiple partially structured states co-exist in the GPCR native ensemble, with the TM helices 1, 6 and 7 displaying varied folding status, and shaping the conformational landscape. Strongly coupled residues are anisotropically distributed, accounting for only 13% of the residues, illustrating that a large number of residues are inherently dynamic. Active-state GPCRs are characterized by reduced conformational heterogeneity with altered coupling-patterns distributed throughout the structural scaffold. In silico alanine-scanning mutagenesis reveals that extra- and intra-cellular faces of GPCRs are coupled thermodynamically, highlighting an exquisite structural specialization and the fluid nature of the intramolecular interaction network. The ensemble-based perturbation methodology presented here lays the foundation for understanding allosteric mechanisms and the effects of disease-causing mutations in GCPRs.

SUBMITTER: Anantakrishnan S

PROVIDER: S-EPMC9829892 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Thermodynamic architecture and conformational plasticity of GPCRs.

Anantakrishnan Sathvik S Naganathan Athi N AN

Nature communications 20230109 1

G-protein-coupled receptors (GPCRs) are ubiquitous integral membrane proteins involved in diverse cellular signaling processes. Here, we carry out a large-scale ensemble thermodynamic study of 45 ligand-free GPCRs employing a structure-based statistical mechanical framework. We find that multiple partially structured states co-exist in the GPCR native ensemble, with the TM helices 1, 6 and 7 displaying varied folding status, and shaping the conformational landscape. Strongly coupled residues are ...[more]

PMID: 36624096

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Project description:Human NFU has been implicated in the formation of inorganic sulfide required for cellular iron-sulfur cluster biosynthesis. The protein contains a well-structured N-terminal domain and a C-terminal domain with molten globule characteristics that also contains a thioredoxin-like pair of redox active Cys residues that promote persulfide reductase activity. Recent reports have highlighted the existence of structural flexibility in the ISU/IscU-type scaffold proteins that mediate Fe-S cluster assembly, which is also likely to serve an important role in the pathway to Fe-S cluster maturation. We have previously reported similar structural mobility for the C-terminal domain of human NFU, a protein that has been implicated in the production of sulfide for cluster synthesis, while homologous proteins have also been suggested to serve as Fe-S cluster carriers. Herein we quantitatively characterize the structural stability of the two domains of human NFU and in particular the functional C-terminal domain. The results of differential scanning calorimetry and variable temperature circular dichroism (VTCD) studies have been used to analyze the temperature-dependent structural melting profiles of the N- and C-terminal domains, relative to both full-length NFU and an equimolar ratio of the N- and C-terminal domains, and correlated with structural information derived from NMR data. Calorimetry results indicate that the C-terminal NFU domain undergoes a significant structural stabilization following interaction with the N-terminal domain, which resulted in a novel and distinctive transition melting profile (Tm(sec) = 58.1 ± 0.4 °C, ?Hv(sec) = 60.4 ± 5.3 kcal/mol, Tm(ter) = 49.3 ± 0.3 °C, ?Hv(ter) = 71.8 ± 5.8 kcal/mol). VTCD experiments also revealed a secondary structure transition at 59.2 °C in agreement with calorimetry results. The degree of stabilization was found to be more significant in the full-length NFU, as the C-terminal domain transitions were recorded at higher temperatures (Tm(sec) = 63.3 ± 3.4 °C, ?Hv(sec) = 41.8 ± 8.2 kcal/mol). The interactions between the two domains demonstrated the hallmarks of a hydrophobic character, as increased ionic strength decreased the degree of stabilization of the C-terminal domain. An increase of 2% in ?-helix content further supports interaction between the two domains, leading to greater secondary structure stabilization. Heteronuclear single-quantum coherence experiments indicate that the C-terminal domain adopts an alternate tertiary conformation following binding to the N-terminal domain. The structural rigidity of the N-terminal domain leads to an alternative conformation of the C-terminal domain, suggesting that such an interaction, although weaker than that of the covalently attached native NFU, is important for the structural chemistry of the native full-length protein. The results also emphasize the likely general importance of such structural flexibility in select proteins mediating metal cofactor biosynthesis.

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Thermodynamic architecture and conformational plasticity of GPCRs.

Publications

Thermodynamic architecture and conformational plasticity of GPCRs.

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