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Abstract: Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information. [Copyright &y& Elsevier]

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)( s Y3, s Y10, s Y14) peptide cross reacts with CXCL12 and with lower K D in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt-receptor peptide binding. The Nt-CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate-binding site by an allovalency mechanism like that observed for CCL5 binding of Nt-CCR5 peptides. Nt-CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV-1 gp120 and interact with all three tyrosine/sulfated tyrosine-binding pockets of CXCL12. We suggest that the chemokine-receptors Nt-segments bind promiscuously to cognate and non-cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine-receptors' Nt-segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study., (© 2023 Federation of European Biochemical Societies.)

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 YU2 with a 27-residue N-terminal CCR5 peptide sulphated at position Y10 and Y14, i.e. Nt-CCR5, were studied using 13 C-edited-HMQC methyl-NOESY [ 1 H( 13 C)- 1 H], 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 gp120 YU2 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., (© 2021 Federation of European Biochemical Societies.)

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., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The N-terminal segment of the chemokine receptor Human CC chemokine receptor 5 (CCR5), Nt-CCR5, contains four tyrosine residues, Y3, Y10, Y14, and Y15. Sulfation of at least two of these tyrosine residues was found to be essential for high-affinity binding of CCR5 to its chemokine ligands. Here, we show that among the monosulfated Nt-CCR5(8-20) peptide surrogates (sNt-CCR5) those sulfated at Y15 and Y14 have the highest affinity for the CC chemokine ligand 5 (CCL5) chemokine in comparison with monosulfation at position Y10. Sulfation at Y3 was not investigated. A peptide sulfated at both Y14 and Y15 has the highest affinity for CCL5 by up to a factor of 3, in comparison with the other disulfated (sNt-CCR5) peptides. Chemical shift perturbation analysis and transferred nuclear Overhauser effect measurements indicate that the sulfated tyrosine residues interact with the same CCL5-binding pocket and that each of the sulfated tyrosines at positions 10, 14, and 15 can occupy individually the binding site on CCL5 in a similar manner, although with somewhat different affinity, suggesting the possibility of allovalency in sulfated Nt-CCR5 peptides. The affinity of the disulfated peptides to CCL5 could be increased by this allovalency and by stronger electrostatic interactions., (© 2020 Federation of European Biochemical Societies.)

Many proteins interact with their ligand proteins by recognition of short linear motifs that are often intrinsically disordered. These interactions are usually weak and are characterized by fast exchange. NMR spectroscopy is a powerful tool to study weak interactions. The methods that have been commonly used are analysis of chemicals shift perturbations (CSP) upon ligand binding and saturation transfer difference spectroscopy. These two methods identify residues at the binding interface between the protein and its ligand. In the present study, we used a combination of transferred-NOE, specific methyl-labeling and an optimized isotope-edited/isotope-filtered NOESY experiment to study specific interactions between the 42 kDa p38α mitogen-activated protein kinase and the kinase interaction motif (KIM) on the STEP phosphatase. These measurements distinguished between residues that both exhibit CSPs upon ligand binding and interact with the KIM peptide from residues that exhibit CSPs but do not interact with the peptide. In addition, these results provide information about pairwise interactions that is important for a more reliable docking of the KIM peptide into its interacting surface on p38α. This combination of techniques should be applicable for many protein-peptide complexes up to 80 kDa for which methyl resonance assignment can be achieved.

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.

Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core YU 2 gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N-terminus of CCR5., (© 2018 Federation of European Biochemical Societies.)

The inflammatory chemokine CCL5, which binds the chemokine receptor CCR5 in a two-step mechanism so as to activate signaling pathways in hematopoetic cells, plays an important role in immune surveillance, inflammation, and development as well as in several immune system pathologies. The recently published crystal structure of CCR5 bound to a high-affinity variant of CCL5 lacks the N-terminal segment of the receptor that is post-translationally sulfated and is known to be important for high-affinity binding. Here, we report the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated peptide corresponding to the missing first 27 residues of CCR5. Our structures show that two sulfated tyrosine residues, sY10 and sY14, as well as the unsulfated Y15 form a network of strong interactions with a groove on a surface of CCL5 that is formed from evolutionarily conserved basic and hydrophobic amino acids. We then use our NMR structures, in combination with available crystal data, to create an atomic model of full-length wild-type CCR5:CCL5. Our findings reveal the structural determinants involved in the recognition of CCL5 by the CCR5 N terminus. These findings, together with existing structural data, provide a complete structural framework with which to understand the specificity of receptor:chemokine interactions., Database: Structural data are available in the PDB under the accession number 6FGP., (© 2018 Federation of European Biochemical Societies.)

We are developing a rapid, time-resolved method using laser-activated cross-linking to capture protein-peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding-yeast mating pheromone (α-factor) and the decapeptide human gonadotropin-releasing hormone (GnRH). Cross-linking of α-factor, using a biotinylated, photoactivatable p-benzoyl-L-phenylalanine (Bpa)-modified analog, was energy-dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA-peptide complex. The cross-linked complex was trypsinized and then interrogated with nano-LC-MS/MS to identify the peptide cross-links. Cross-linking was greatly facilitated by Bpa in the peptide, but some cross-linking occurred at higher laser powers and high concentrations of a non-Bpa-modified α-factor. This was supported by experiments using GnRH, a peptide with sequence homology to α-factor, which was likewise found to be cross-linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α-factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser-activation to facilitate cross-linking of Bpa-containing molecules to proteins. The rapid cross-linking procedure and high performance of MS/MS to identify cross-links provides a method to interrogate protein-peptide interactions in a living cell in a time-resolved manner., (Copyright © 2017 John Wiley & Sons, Ltd.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The Saccharomyces cerevisiae α-factor receptor Ste2p has been used extensively as a model to understand the molecular mechanism of signal transduction by G protein-coupled receptors (GPCRs). Single and double cysteine mutants of Ste2p were created and served as surrogates to detect intramolecular interactions and dimerization of Ste2p using disulfide cross-linking methodology. When a mutation was introduced into the phylogenetically conserved tyrosine residue at position 26 (Y26C) in the N-terminus of Ste2p, dimerization was increased greatly. The amount of dimer formed by this Y26C mutant was greatly reduced by ligand binding even though the ligand binding site is far removed from the N-terminus; the lowering of the dimer formation was consistent with a conformational change in the N-terminus of the receptor upon activation. Dimerization was decreased by double mutations Y26C/V109C or Y26C/T114C indicating that Y26 is in close proximity to V109 and T114 of extracellular loop 1 in native Ste2p. Combined with earlier studies, these results indicate previously unrecognized roles for the N-terminus of Ste2p, and perhaps of GPCRs in general, and reveal a specific N-terminus residue or region, that is involved in GPCR signaling, intrareceptor interactions, and receptor dimerization., (Copyright © 2017 Elsevier B.V. All rights reserved.)

NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (K D > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the 13 C-edited/ 13 C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the 13 C-edited/ 13 C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1-27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the 13 C-edited/ 13 C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1-27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1-27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex., (© 2017 Federation of European Biochemical Societies.)

V3-directed antibodies are present in practically all HIV-1 infected patients and in individuals vaccinated with gp120. The levels of maternal V3-directed antibodies were recently shown to correlate with reduced mother to child transmission, and V3 IgGs were found to be a negative correlate of risk in the RV-144 human trial. mAb directed to the tip of the V3 are capable of broad neutralization of Tier-1 and some Tier-2 viruses. Here we report an immunofocusing approach using conformationally constrained V3 peptides of different lengths. Immunofocusing with short constrained V3 peptides following immunizations with long constrained V3 peptides resulted in sera with improved neutralization of Tier-1B viruses in comparison with immunizations with the long constrained peptide alone. Immunizations only with the short constrained peptide were ineffective. Our results demonstrate that immunofocusing with constrained V3 peptides of different lengths could improve the induction of HIV-1 neutralizing antibodies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031-4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187-3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae . The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.

We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing S. cerevisiae with the target microbe and modifying growth conditions accordingly., Background: Di-/tripeptides are one of the major sources of nitrogen, carbon, and amino acids for all organisms. Synthetic peptides containing a toxic amino acid residue provide an experimental approach to measure peptide transport and/or utilization in Saccharomyces cerevisiae . Hydrolysis of internalized peptides by intracellular peptidases or proteases releases the toxic residue leading to an easily detectable zone (halo) of growth arrest on a lawn of cells plated in a Petri plate. For example, upon intracellular hydrolysis the toxic peptide Ala-Eth releases ethionine (Eth), a methionine antagonist which interferes with the incorporation of amino acids into proteins and with the normal methylation of DNA and other methylation pathways, thereby leading to cell death. When spotted onto a lawn of yeast cells, the transported dipeptide Ala-Eth will inhibit growth, and a clear 'halo' will form in the lawn of cells around the region where the Eth-containing toxic peptide is spotted (Figure 1A). The assay described here for determination of peptide toxicity in S. cerevisiae may be generalized as follows: (1) it may be modified to determine toxicity of any substrate by simply using a putative toxic compound in place of a peptide containing a toxic amino acid, or (2) it may be modified to determine toxicity of a substrate toward any microorganism by replacing S. cerevisiae in the assay with the target organism. It is a simple, inexpensive and relatively rapid method for determining substrate toxicities as modified for the specific organism and toxic moiety assayed.

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Intermolecular NOE interactions are invaluable for structure determination of biomolecular complexes by NMR and they represent the "gold-standard" amongst NMR measurements for characterizing interfaces. These NOEs constitute only a small fraction of the observed NOEs in a complex and are usually weaker than many of the intramolecular NOEs. A number of methods have been developed to remove the intramolecular NOEs that interfere with the identification of intermolecular NOEs. NMR experiments used to observe intermolecular NOE interactions in large protein complexes must cope with the short T2 relaxation time of the protons and heteronuclei in these complexes because they result in severe losses in sensitivity. The isotope-edited/isotope-filtered experiment is a powerful method for extraction of intermolecular NOEs in biomolecular complexes. Its application to large protein complexes is limited because of severe losses in signal-to-noise ratio caused by delays in the pulse sequence necessary for the multiple magnetization transfer steps between protons and heteronuclei. Isotope-edited/isotope-edited experiments, in which one protein is usually labeled with 13 C and the other is labeled with 15 N, reduce possible artifacts in the filtering experiments and improve somewhat the sensitivity of these experiments. Sensitivity can also be improved by deuteration of the components of the complex in order to replace either or both of the filtering or editing steps. Asymmetric deuteration, where aromatic residues in one protein and non-aromatic amino acids in the other are reverse protonated, can eliminate the editing and the filtering steps altogether, thus maintaining high sensitivity even for large proteins complexes. Difference spectroscopy and the use of 2D NOESY experiments without using editing or filtering steps can significantly increase the signal-to-noise ratio in experiments aimed at observing intermolecular NOEs. The measurement of NOESY spectra of three different preparations of a heterodimeric complex under investigation in which one or neither of the components is uniformly deuterated, and calculation of a double difference spectrum provides information on all intermolecular NOEs of non-exchangeable protons. Recent studies indicate that many protein-protein interactions are actually between a protein and a linear peptide recognition motif of the second protein, and determinants represented by linear peptides contribute significantly to the binding energy. NMR is a very versatile method to study peptide-protein interactions over a wide range of binding affinities and binding kinetics. Protein-peptide interactions in complexes exhibiting tight binding can be studied using single and/or multiple deuteration of the peptide residues and measuring a difference NOESY spectrum. This difference spectrum will show exclusively intra- and intermolecular interactions of the peptide protons that were deuterated. Transferred nuclear Overhauser spectroscopy (TRNOE) extends NMR to determine interactions within and between a weakly-bound rapidly-exchanging peptide and its protein target. TRNOE, together with asymmetric deuteration, is applicable to complexes up to ∼100KDa and is highly sensitive, taking advantage of the long average T2 of the peptide protons. Among the methods described in this review, TRNOE has the best potential to determine intermolecular NOEs for the upper molecular weight limit of proteins that can be studied in detail by NMR., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Weak protein-protein and protein-ligand interactions play important roles in biological recognition. In many cases, simplification of structural studies of large protein complexes is achieved by investigation of the interaction between the protein and a weakly binding segment of its protein ligand. Detection of pairwise interactions in such complexes is a major challenge for both X-ray crystallography and nuclear magnetic resonance. We demonstrate that transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Using this approach, we revealed interactions between tyrosine residues of a 27-residue peptide (deuterated at Ile and Val residues) corresponding to the N-terminal segment of the human C-C chemokine receptor 5 (CCR5) chemokine receptor, and a 43 kDa construct of gp120 envelope protein of human immunodeficiency virus type 1 (deuterated on all aromatics) complexed with a cluster of differentiation 4-mimic miniprotein. The complex was present mostly as a dimer as determined by T2 relaxation measurements. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons, predominantly of isoleucine residues, and also of leucine and/or valine residues of gp120. The TRNOE/asymmetric deuteration method benefits from the sensitivity of the homonuclear NOESY experiment and does not suffer the sensitivity losses associated with isotope-edited/isotope-filtered approaches that rely on magnetization transfer between protons and heteronuclei that are bonded to them. The technique can be widely applied for studying large protein complexes that exhibit fast off-rates., (© 2016 Federation of European Biochemical Societies.)

G protein-coupled receptors (GPCRs) are found in all eukaryotic cells examined to date where they function as membrane-bound proteins that bind a multitude of extracellular ligands to initiate intracellular signal transduction systems controlling cellular physiology. GPCRs have seven heptahelical membrane spanning domains connected by extracellular and intracellular loops with an extracellular N-terminus and an intracellular C-terminus. The N-terminus has been the least studied domain of most GPCRs. The yeast Ste2p protein, the receptor for the thirteen amino acid peptide pheromone α-factor, has been used extensively as a model to study GPCR structure and function. In this study we constructed a number of deletions of the Ste2p N-terminus and uncovered an unexpected function as a negative regulatory domain. We examined the role of the N-terminus in expression, signaling function and ligand-binding properties and found that the residues 11-30 play a critical role in receptor expression on the cell surface. The studies also indicated that residues 2-10 of the N-terminus are involved in negative regulation of signaling as shown by the observation that deletion of these residues enhanced mating and gene induction. Furthermore, our results indicated that the residues 21-30 are essential for optimal signaling. Overall, we propose that the N-terminus of Ste2p plays multiple regulatory roles in controlling receptor function., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors., Database: The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505., (© 2015 FEBS.)

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The isolation of mutations affecting the stabilities of transmembrane proteins is useful for enhancing the suitability of proteins for structural characterization and identification of determinants of membrane protein stability. We have pursued a strategy for the identification of stabilized variants of the yeast α-factor receptor Ste2p. Because it was not possible to screen directly for mutations providing thermal stabilization, we first isolated a battery of destabilized temperature-sensitive variants, based on loss of signaling function and decreased levels of binding of the fluorescent ligand, and then screened for intragenic second-site suppressors of these phenotypes. The initial screens recovered singly and multiply substituted mutations conferring temperature sensitivity throughout the predicted transmembrane helices of the receptor. All of the singly substituted variants exhibit decreases in cell-surface expression. We then screened randomly mutagenized libraries of clones expressing temperature-sensitive variants for second-site suppressors that restore elevated levels of binding sites for fluorescent ligand. To determine whether any of these were global suppressors, and thus likely stabilizing mutations, they were combined with different temperature-sensitive mutations. Eight of the suppressors exhibited the ability to reverse the defect in ligand binding of multiple temperature-sensitive mutations. Combining certain suppressors into a single allele resulted in levels of suppression greater than that seen with either suppressor alone. Solubilized receptors containing suppressor mutations in the absence of temperature-sensitive mutations exhibit a reduced tendency to aggregate during immobilization on an affinity matrix. Several of the suppressors also exhibit allele-specific behavior indicative of specific intramolecular interactions in the receptor.

This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from Escherichia coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast G protein-coupled receptor can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

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., (© 2015 Elsevier Inc. All rights reserved.)

The envelope spike of HIV-1, which consists of three external gp120 and three transmembrane gp41 glycoproteins, recognizes its target cells by successively binding to its primary CD4 receptor and a coreceptor molecule. Until recently, atomic-resolution structures were available primarily for monomeric HIV-1 gp120, in which the V1, V2 and V3 variable loops were omitted (gp120core ), in complex with soluble CD4 (sCD4). Differences between the structure of HIV gp120core in complex with sCD4 and the structure of unliganded simian immunodeficiency virus gp120core led to the hypothesis that gp120 undergoes a major conformational change upon sCD4 binding. To investigate the conformational flexibility of gp120, we generated two forms of mutated gp120 amenable for NMR studies: one with V1, V2 and V3 omitted ((mut) gp120core ) and the other containing the V3 region [(mut) gp120core (+V3)]. The TROSY-(1)H-(15)N-HSQC spectra of [(2)H, (13)C, (15)N]Arg-labeled and [(2)H, (13)C, (15)N]Ile-labeled unliganded (mut) gp120core showed many fewer crosspeaks than the expected number, and also many fewer crosspeaks in comparison with the labeled (mut) gp120core bound to the CD4-mimic peptide, CD4M33. This finding suggests that in the unliganded form, (mut) gp120core shows considerable flexibility and motions on the millisecond time scale. In contrast, most of the expected crosspeaks were observed for the unliganded (mut) gp120core (+V3), and only a few changes in chemical shift were observed upon CD4M33 binding. These results indicate that (mut) gp120core (+V3) does not show any significant conformational flexibility in its unliganded form and does not undergo any significant conformational change upon CD4M33 binding, underlining the importance of V3 in stabilizing the gp120core conformation., (© 2014 FEBS.)

The structural characterization of G protein-coupled receptors has surged since the development of methodologies to facilitate the crystallization of these highly helical, seven transmembrane, integral membrane receptors. In the past seven years, eighteen GPCR structures were determined by X-ray crystallography. The crystal structures represent a static picture of these conformationally flexible signal transducers. Analyses that probe their dynamics and conformational changes require other techniques, in particular solution state nuclear magnetic resonance studies. Such investigations are challenged by the size of GPCRs, their α-helical structure, which limits resonance dispersion, their tendencies to aggregate in micellar preparations and their conformational heterogeneity. For many years, groups have been studying GPCR fragments as a means to overcome some of these difficulties. The results of these fragment analyses are presented here. Review of the literature reveals that much of the original work depended on circular dichroism, infra-red spectroscopy and fluorescence approaches. High resolution structures obtained by NMR are compared, where applicable, to the available crystal structures. In most cases, the work done on fragments by biophysical analysis is validated by these comparisons. Our perspective on the field of GPCR fragment analysis is presented together with the future goals that must be considered if work with fragments is continued., (© 2014 Wiley Periodicals, Inc.)