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Article ; Online: A Paradigm for Peptide Hormone-GPCR Analyses.

Naider, Fred / Becker, Jeffrey M

2020 Volume 25, Issue 18

Abstract: Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the ... ...

Abstract	Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast
MeSH term(s)	Allosteric Regulation ; Binding Sites ; Ligands ; Microscopy, Fluorescence ; Peptide Hormones/chemistry ; Peptide Hormones/metabolism ; Protein Binding ; Protein Domains ; Receptors, G-Protein-Coupled/chemistry ; Receptors, G-Protein-Coupled/genetics ; Receptors, G-Protein-Coupled/metabolism ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Ligands ; Peptide Hormones ; Receptors, G-Protein-Coupled
Language	English
Publishing date	2020-09-18
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules25184272
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Article ; Online: The chemokines CCL5 and CXCL12 exhibit high-affinity binding to N-terminal peptides of the non-cognate receptors CXCR4 and CCR5, respectively.

Kessler, Naama / Akabayov, Sabine R / Cohen, Leah S / Scherf, Tali / Naider, Fred / Anglister, Jacob

The FEBS journal

2023 Volume 291, Issue 3, Page(s) 458–476

Abstract: CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. ...

Abstract	CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N-terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt-CCR5(8-20) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt-CCR5(1-27)(
MeSH term(s)	Chemokine CCL5/chemistry ; Receptors, CXCR4/metabolism ; Receptors, CCR5/chemistry ; Chemokine CXCL12 ; Peptides/chemistry ; Tyrosine
Chemical Substances	Chemokine CCL5 ; Receptors, CXCR4 ; Receptors, CCR5 ; Chemokine CXCL12 ; Peptides ; Tyrosine (42HK56048U)
Language	English
Publishing date	2023-12-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.17013
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Article ; Online: A Paradigm for Peptide Hormone-GPCR Analyses

Fred Naider / Jeffrey M. Becker

Molecules, Vol 25, Iss 4272, p

2020 Volume 4272

Abstract	Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.
Keywords	peptide pheromone ; G protein-coupled receptors ; nuclear magnetic resonance ; photoactivated crosslinking ; chemical crosslinking ; receptor mutation ; Organic chemistry ; QD241-441
Subject code	540
Language	English
Publishing date	2020-09-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The Synthesis of Sulfated CCR5 Peptide Surrogates and their Use to Study Receptor-Ligand Interactions.

Naider, Fred / Anglister, Jacob

Protein and peptide letters

2018 Volume 25, Issue 12, Page(s) 1124–1136

Abstract: Background: Tyrosine sulfation is an important post-translational modification of secreted and membrane proteins in multi-cellular organisms. This modification is catalyzed by tyrosylprotein sulfotransferases that often modify tyrosine residues in their ...

Abstract	Background: Tyrosine sulfation is an important post-translational modification of secreted and membrane proteins in multi-cellular organisms. This modification is catalyzed by tyrosylprotein sulfotransferases that often modify tyrosine residues in their target substrates in a heterogeneous manner. Chemokine receptors such as CCR5, which play roles in inflammation, immunity and viral infection, are sulfated on tyrosine residues in their extracellular N-termini. The heterogeneity of the sulfation has made it difficult to obtain atomic-resolution information on this region of CCR5. Homogeneously sulfated peptide surrogates can be efficiently synthesized by chemical and biochemical approaches. This communication reviews current chemical and biochemical methods for peptide tyrosine sulfation and the use of N-terminal CCR5 peptide surrogates in biochemical and structural analyses. Conclusion: Using solid phase peptide synthesis and synthons containing sulfotyrosine or sulfotyrosine neopentyl esters peptides containing up to 30 residues with multiple sulfotyrosines can be synthesized and purified in high (>50-70%) yield. Such peptides can be isotopically labeled at selected positions and used in detailed NMR investigations to investigate the interactions of sulfotyrosine residues with receptors. The application of transferred NOE studies to investigate CCL5/CCR5 interactions has led to the determination of pairwise interactions between the chemokine and its receptor.
MeSH term(s)	Animals ; Humans ; Ligands ; Models, Molecular ; Peptides/chemical synthesis ; Peptides/chemistry ; Peptides/pharmacology ; Protein Binding/drug effects ; Protein Conformation ; Receptors, CCR5/chemistry ; Receptors, CCR5/metabolism ; Solid-Phase Synthesis Techniques/methods ; Tyrosine/analogs & derivatives ; Tyrosine/chemistry
Chemical Substances	CCR5 protein, human ; Ligands ; Peptides ; Receptors, CCR5 ; tyrosine O-sulfate (29166358BF) ; Tyrosine (42HK56048U)
Language	English
Publishing date	2018-10-31
Publishing country	Netherlands
Document type	Journal Article ; Review
ZDB-ID	1280776-x
ISSN	1875-5305 ; 0929-8665
ISSN (online)	1875-5305
ISSN	0929-8665
DOI	10.2174/0929866525666181101103834
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Article ; Online: Oligomerization of yeast α-factor receptor detected by fluorescent energy transfer between ligands.

Connelly, Sara M / Sridharan, Rajashri / Naider, Fred / Dumont, Mark E

Biophysical journal

2021 Volume 120, Issue 22, Page(s) 5090–5106

Abstract: G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive ... ...

Abstract	G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily. A novel, to our knowledge, procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently labeled derivatives of the receptor agonist α-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently labeled derivatives of α-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. Although the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native α-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane.
MeSH term(s)	Fluorescence Resonance Energy Transfer ; Ligands ; Receptors, G-Protein-Coupled ; Receptors, Mating Factor/genetics ; Saccharomyces cerevisiae
Chemical Substances	Ligands ; Receptors, G-Protein-Coupled ; Receptors, Mating Factor
Language	English
Publishing date	2021-10-08
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2021.10.005
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Article ; Online: Multiple binding modes of an N-terminal CCR5-peptide in complex with HIV-1 gp120.

Moseri, Adi / Akabayov, Sabine R / Cohen, Leah S / Naider, Fred / Anglister, Jacob

The FEBS journal

2021 Volume 289, Issue 11, Page(s) 3132–3147

Abstract: The N-terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high-affinity binding to gp120. In the present study, the interactions of ... ...

Abstract	The N-terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high-affinity binding to gp120. In the present study, the interactions of gp120
MeSH term(s)	HIV Envelope Protein gp120/genetics ; HIV Envelope Protein gp120/metabolism ; HIV-1/metabolism ; Peptides/chemistry ; Protein Binding ; Protons ; Receptors, CCR5/chemistry ; Tyrosine/metabolism
Chemical Substances	HIV Envelope Protein gp120 ; Peptides ; Protons ; Receptors, CCR5 ; Tyrosine (42HK56048U)
Language	English
Publishing date	2021-12-28
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.16328
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Article: Multiple binding modes of an N‐terminal CCR5‐peptide in complex with HIV‐1 gp120

Moseri, Adi / Akabayov, Sabine R. / Cohen, Leah S. / Naider, Fred / Anglister, Jacob

FEBS journal. 2022 June, v. 289, no. 11

2022

Abstract: The N‐terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high‐affinity binding to gp120. In the present study, the interactions of gp120YU₂ with a 27‐residue N‐terminal CCR5 peptide sulphated at position ...

Abstract	The N‐terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high‐affinity binding to gp120. In the present study, the interactions of gp120YU₂ with a 27‐residue N‐terminal CCR5 peptide sulphated at position Y10 and Y14, i.e. Nt‐CCR5, were studied using ¹³C‐edited‐HMQC methyl‐NOESY [¹H(¹³C)‐¹H], combined with transferred NOE NMR spectroscopy. A large number of pairwise interactions were observed between the methyl protons of methionine, threonine, valine and isoleucine residues of gp120, and the aromatic tyrosine‐protons of Nt‐CCR5. M434, V120 and V200 of gp120 were found to interact with all four tyrosine residues, Y3, sY10, sY14 and Y15. Particularly intriguing was the observation that Y3 and Y15 interact with the same gp120 methyl protons. Such interactions cannot be explained by the single cryo‐EM structure of gp120/CD4/CCR5 complex published recently (Nature, 565, 318–323, 2019). Rather, they are consistent with the existence of a dynamic equilibrium involving two or more binding modes of Nt‐CCR5 to gp120. These different modes of binding can coexist because the surface of gp120 contains two sites that can optimally interact with a sulphated tyrosine residue and two sites that can interact favorably with a non‐sulphated tyrosine residue. Modelling of gp120YU₂ complexed with the Nt‐CCR5 peptide or with the entire CCR5 receptor provides an explanation for the NMR observations and the existence of these different binding modes of the disordered N‐terminus of CCR5. The data presented extend our understanding of the two‐step model and suggest a more variable binding mode of Nt‐CCR5 with gp120.
Keywords	CCR5 receptor ; isoleucine ; methionine ; models ; nuclear magnetic resonance spectroscopy ; peptides ; threonine ; tyrosine ; valine
Language	English
Dates of publication	2022-06
Size	p. 3132-3147.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.16328
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Article ; Online: Cross-linking strategies to study peptide ligand-receptor interactions.

Becker, Jeffrey M / Naider, Fred

Methods in enzymology

2015 Volume 556, Page(s) 527–547

Abstract: Experiments are described that allowed cross-linking of analogs of a 13-amino acid peptide into the binding site of a model G protein-coupled receptor. Syntheses of peptide analogs that were used for photochemical or chemical cross-linking were carried ... ...

Abstract	Experiments are described that allowed cross-linking of analogs of a 13-amino acid peptide into the binding site of a model G protein-coupled receptor. Syntheses of peptide analogs that were used for photochemical or chemical cross-linking were carried out using solid-phase peptide synthesis. Chemical cross-linking utilized 3,4-dihydroxy-l-phenylalanine-incorporated peptides and subsequent periodate-mediated activation, whereas photochemical cross-linking was mediated by p-benzoyl-l-phenylalanine (Bpa)-labeled peptides and UV-initiated activation. Mass spectrometry was employed to locate the site(s) in the receptor that formed the cross-links to the ligand. We also describe a method called unnatural amino acid replacement that allowed capture of a peptide ligand into the receptor. In this method, the receptor was genetically modified by replacement of a natural amino acid with Bpa. The modified receptor was UV-irradiated to capture the ligand. The approaches described are applicable to other peptide-binding proteins and can reveal the ligand-binding site in atomic detail.
MeSH term(s)	Amino Acid Sequence ; Animals ; Benzophenones/chemistry ; Cross-Linking Reagents/chemistry ; Humans ; Ligands ; Mass Spectrometry/methods ; Molecular Sequence Data ; Peptides/chemistry ; Peptides/metabolism ; Phenylalanine/analogs & derivatives ; Phenylalanine/chemistry ; Receptors, G-Protein-Coupled/chemistry ; Receptors, G-Protein-Coupled/metabolism ; Solid-Phase Synthesis Techniques ; Ultraviolet Rays
Chemical Substances	1,4-dihydrophenylalanine ; 4-benzoylphenylalanine ; Benzophenones ; Cross-Linking Reagents ; Ligands ; Peptides ; Receptors, G-Protein-Coupled ; Phenylalanine (47E5O17Y3R)
Language	English
Publishing date	2015
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1557-7988 ; 0076-6879
ISSN (online)	1557-7988
ISSN	0076-6879
DOI	10.1016/bs.mie.2014.12.001
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Article: Synthesis, biosynthesis, and characterization of transmembrane domains of a G protein-coupled receptor.

Naider, Fred

Methods in molecular biology (Clifton, N.J.)

2007 Volume 386, Page(s) 95–121

Abstract: Peptide fragments have been widely used in biophysical studies on specific regions of integral membrane proteins. Because of their inherent insoluble nature and tendency to aggregate the preparation of such model peptides is challenging. We have ... ...

Abstract	Peptide fragments have been widely used in biophysical studies on specific regions of integral membrane proteins. Because of their inherent insoluble nature and tendency to aggregate the preparation of such model peptides is challenging. We have developed synthetic and biosynthetic approaches to prepare peptides containing single and multiple domains of a G protein-coupled receptor. Both the synthetic and biosynthetic products can be isolated by reversed-phase high-performance liquid chromatography to near homogeneity. The biosynthetic product, a fusion protein, is processed by CNBr cleavage to yield the target peptide in various isotopic forms. The final peptides are studied by circular dichroism spectroscopy to determine their secondary structure under a variety of conditions.
MeSH term(s)	Amino Acid Sequence ; Chromatography, High Pressure Liquid/methods ; Circular Dichroism ; Cyanogen Bromide ; Drug Design ; Electrophoresis, Polyacrylamide Gel/methods ; Escherichia coli/genetics ; Models, Molecular ; Molecular Biology/methods ; Molecular Sequence Data ; Peptide Fragments/biosynthesis ; Peptide Fragments/chemical synthesis ; Peptide Fragments/chemistry ; Peptide Fragments/genetics ; Plasmids/genetics ; Protein Engineering ; Protein Structure, Tertiary ; Receptors, G-Protein-Coupled/biosynthesis ; Receptors, G-Protein-Coupled/chemistry ; Receptors, G-Protein-Coupled/genetics ; Receptors, Mating Factor/biosynthesis ; Receptors, Mating Factor/chemistry ; Receptors, Mating Factor/genetics ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Saccharomyces cerevisiae Proteins/biosynthesis ; Saccharomyces cerevisiae Proteins/chemical synthesis ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/genetics
Chemical Substances	Peptide Fragments ; Receptors, G-Protein-Coupled ; Receptors, Mating Factor ; Recombinant Fusion Proteins ; STE2 protein, S cerevisiae ; Saccharomyces cerevisiae Proteins ; Cyanogen Bromide (OS382OHJ8P)
Language	English
Publishing date	2007
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1064-3745
ISSN	1064-3745
DOI	10.1007/978-1-59745-430-8_4
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Article: Uptake Assay for Radiolabeled Peptides in Yeast.

Hauser, Melinda / Cai, Houjian / Naider, Fred / Becker, Jeffrey M

Bio-protocol

2017 Volume 6, Issue 22

Abstract: We describe an assay for measuring the uptake of radioactive peptides into the ... ...

Abstract	We describe an assay for measuring the uptake of radioactive peptides into the yeast
Language	English
Publishing date	2017-11-02
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325
ISSN	2331-8325
DOI	10.21769/BioProtoc.2026
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