Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits

Park, Jihye; Selvam, Balaji; Sanematsu, Keisuke; Shigemura, Noriatsu; Shukla, Diwakar; Procko, Erik

doi:10.1074/jbc.ra118.006173

Cited by 56 publications

(88 citation statements)

References 77 publications

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“…117 residues within the protease domain of ACE2 were diversified by overlap extension PCR ( 35 ) using primers with degenerate NNK codons. The plasmid library was transfected in to Expi293F cells using Expifectamine under conditions previously shown to typically give no more than a single coding variant per cell ( 30, 31 ); 1 ng coding plasmid was diluted with 1,500 ng pCEP4-ΔCMV carrier plasmid per ml of cell culture at 2 × 10 6 / ml, and the medium was replaced 2 h post-transfection. The cells were collected after 24 h, washed with ice-cold PBS-BSA, and incubated for 30 minutes on ice with a 1/20 (replicate 1) or 1/40 (replicate 2) dilution of medium containing RBD-sfGFP into PBS supplemented with 0.2 % bovine serum albumin (PBS-BSA).…”

Section: Methodsmentioning

confidence: 99%

“…The ACE2 library was transiently expressed in human Expi293F cells under conditions that typically yield no more than one coding variant per cell, providing a tight link between genotype and phenotype ( 30, 31 ). Cells were then incubated with a subsaturating dilution of medium containing the RBD of SARS-CoV-2 fused C-terminally to superfolder GFP (sfGFP: ( 32 )) (Fig.…”

Section: Figurementioning

confidence: 99%

“…This indicates that most, but not all, nonsynonymous mutations in ACE2 did not eliminate surface expression. The library is biased towards solvent-exposed residues and has few substitutions of buried hydrophobics that might have bigger effects on plasma membrane trafficking ( 31 ).…”

Section: Figurementioning

confidence: 99%

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The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2

Procko

2020

Preprint

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133

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The rapid and escalating spread of SARS coronavirus 2 (SARS-CoV-2) 6 poses an immediate public health emergency. The viral spike protein S binds ACE2 7 on host cells to initiate molecular events that release the viral genome 8intracellularly. Soluble ACE2 inhibits entry of both SARS and SARS-2 coronaviruses 9 by acting as a decoy for S binding sites, and is a candidate for therapeutic and 10 prophylactic development. Using deep mutagenesis, variants of ACE2 are identified 11with increased binding to the receptor binding domain of S. Mutations are found 12 across the interface, in the N90-glycosylation motif, and at buried sites where they 13 are predicted to enhance folding and presentation of the interaction epitope. When 14 single substitutions are combined, large increases in binding can be achieved. The 15 mutational landscape offers a blueprint for engineering high affinity proteins and 16peptides that block receptor binding sites on S to meet this unprecedented 17 challenge.

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Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2

Procko

2020

Preprint

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133

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“…The library covered 3,524 of 3,600 possible single amino acid substitutions based on a minimum frequency of 2  10 -6 . Expi293F cells at 2  10 6 cells/mL were transfected using Expifectamine (ThermoFisher) with 1 ng library DNA diluted with 1.5 g of pCEP4-CMV (74). The media was replaced 2 hours post transfection.…”

Section: Bimolecular Fluorescence Complementation Expi293f Cells Atmentioning

confidence: 99%

“…Reviewers can access the private data using token: cdktmweknpspvsp Confocal Microscopy. Performed as described in (74). Briefly, transfected HEK293T cells were permeabilized and fixed using the BD Cytofix/Cytoperm kit, and co-stained with anti-FLAG-Cy3 (Sigma-Aldrich) and anti-GM130-Alexa Fluor 488 (ThermoFisher Scientific) or anticalnexin-Alexa Fluor 488 (ThermoFisher Scientific).…”

Section: Bimolecular Fluorescence Complementation Expi293f Cells Atmentioning

confidence: 99%

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

McShan¹,

Devlin²,

Overall³

et al. 2019

Preprint

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Highlights Deep mutagenesis identifies a conformational disulfide-linked epitope as the main requirement for association of nascent MHC-I molecules with the TAPBPR chaperone  Analysis of μs-ms timescale conformational dynamics by methyl NMR reveals allelespecific profiles at the TAPBPR interaction surfaces of peptide-loaded MHC-I molecules  μs-ms dynamics dictate the specificity of TAPBPR interactions for different MHC-I alleles through the sampling of a minor, "excited state" conformation  Restriction of dynamics though an engineered disulfide bond abrogates interactions with TAPBPR, both in solution and on a cellular membrane Keywords Major Histocompatibility Complex • MHC-I • NMR spectroscopy • protein dynamics • molecular chaperone • TAPBPR • peptide editing • deep mutagenesis mapping • conformational landscape • peptide repertoire Graphical Abstract AbstractThe interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance.Here, we demonstrate that the molecular chaperone TAPBPR associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized towards one side of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α 2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α 1 /α 2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of i) chaperoning unstable MHC-I molecules at early stages of their folding process, akin to a holdase with broad allelespecificity, and ii) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, towards editing Sithe repertoire of displayed antigens. Significance StatementThe human population contains thousands of MHC-I alleles, showing a range of dependencies on molecular chaperones for loading of their peptide cargo, which are then displayed on the cell surface for T cell surveillance. Using the chaperone TAPBPR as a model, we combine deep mutagenesis with functional and biophysical data, especially solution NMR, to provide a compl...

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Evolutionary history of ATP‐binding cassette proteins

Srikant

2020

FEBS Letters

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ATP‐binding cassette (ABC) proteins are found in every sequenced genome and evolved deep in the phylogenetic tree of life. ABC proteins form one of the largest homologous protein families, with most being involved in substrate transport across biological membranes, and a few cytoplasmic members regulating in essential processes like translation. The predominant ABC protein classification scheme is derived from human members, but the increasing number of fully sequenced genomes permits to reevaluate this paradigm in the light of the evolutionary history the ABC‐protein superfamily. As we study the diversity of substrates, mechanisms, and physiological roles of ABC proteins, knowledge of the evolutionary relationships highlights similarities and differences that can be attributed to specific branches in protein divergence. While alignments and trees built on natural sequence variation account for the evolutionary divergence of ABC proteins, high‐throughput experiments and next‐generation sequencing creating experimental sequence variation are instrumental in identifying functional constraints. The combination of natural and experimentally produced sequence variation allows a broader and more rational study of the function and physiological roles of ABC proteins.

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Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits

Cited by 56 publications

References 77 publications

The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2

The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

Evolutionary history of ATP‐binding cassette proteins

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