Voltage gated sodium channels (VGSC) are membrane proteins that are important in the central and peripheral nervous systems and cardiac muscles among others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, and several ailments that could lead to sudden cardiac death. Furthermore, these channels are one of the primary targets of toxins from venomous animals. These toxins have been used as an excellent probe to study the function of VGSC due to their specific and potent effect on VGSC. The sea anemone toxin anthopleurin-A (ApA) inhibits the fast inactivation of the cardiac channel NaV1.5 by binding to the S3b-S4a motif (paddle motif) on the voltage sensing domain (VSD) of DIV. Here we used NMR spectroscopy to determine the solution structures of NaV1.5 DIV paddle motif and of ApA toxin, both in DPC micelles. The structure of the paddle motif takes a helix-turn-helix motif that aligns well with the cryo-EM structure of the full channel. Through paramagnetic relaxation enhancement, we determined that the paddle motif is mainly interacting with the interface region of the micelle. Based on 15N NMR relaxation studies, the paddle motif is mostly rigid with some residues at and around the loop in addition to the C-terminus undergoing ps-ns motion. NMR titration experiments between a 15N labeled paddle motif with unlabeled ApA toxin uncovered specific residues on the paddle that likely interact with the toxin. Further electrophysiology studies are needed to confirm the importance of these residues in toxin binding. We are in the process of determining the three-dimensional structure of the toxin-paddle complex in micelle.

ABSTRACTVoltage-gated sodium channels are membrane proteins that play an important role in the propagation of electrical signals by mediating the rising phase of an action potential. Numerous diseases, including epilepsy, extreme pain, and certain cardiac arrhythmias have been linked to defects in these channels. The S3b-S4a helix-turn-helix motif (paddle motif) is a region of the channel that is involved in voltage sensing and undergoes significant structural changes during gating. It is also the binding site for many gating-modifier toxins. We determined the solution structure of the paddle motif from the fourth repeat of NaV1.5 in dodecylphosphocholine micelles by NMR spectroscopy and investigated its dynamics and micelle interactions. The structure displays a helix hairpin with a short connecting loop, and likely represents the activated conformation with three of the first four gating charges facing away from S3. Furthermore, paramagnetic relaxation measurements show that the paddle motif is mainly interacting with the interface region of the micelle. NMR relaxation studies reveal that the paddle motif is mostly rigid, with some residues around the loop region and the last 4 residues on the C-terminus displaying heightened mobility. The structural findings reported here allowed the interpretation of three disease-causing mutations in this region of the human cardiac sodium channel, S1609W, F1617del and T1620M. The establishment of this model system for NMR studies of the paddle region offers a promising platform for future toxin interaction studies in the cardiac sodium channels, and similar approaches may be applied to other sodium channel isoforms.

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 YU2 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.

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.DatabaseStructural data are available in the PDB under the accession number 6FGP

The effects of various lipid bound paramagnetic metal ions on liposomes prepared in the presence of trehalose and chelator lipids are evaluated to observe site-specific signal changes on liposome samples with optimal resolution in solid-state NMR spectroscopy. We found that Mn2+, Gd3+ and Dy3+ have different influences on the lipid 13C sites depending on their penetration depths into the bilayer, which can be extracted as distance information. The trehalose-liposome mixture is efficiently packed into solid-state NMR rotors and provides optimal resolution at reasonable instrument temperatures (10-50 °C). The effectiveness and convenience of the trehalose preparation for studying a membrane protein in liposomes are demonstrated by a membrane sample with a model membrane peptide to show that trehalose is useful to prepare consistent and stable membrane protein liposome samples for solid-state NMR.

Beta-pompilidotoxin (β-PMTX) is a spider wasp toxin which binds on neurotoxin receptor site 3 of neuronal sodium channels. Even though it lacks disulfide bonds and has no structural homology to the α-scorpion toxins and sea anemone toxins that bind at the same site, β-PMTX not only slows inactivation of voltage-gated sodium channels, it also has a high affinity for Nav1.2, but does not affect Nav1.5. Due to its high specificity for Nav1.2 DIV, structural analysis of this 13 amino acid peptide may prove vital in understanding gating mechanism of the channel, because it potentially allows for the channel protein to be "locked" into a specific gating state, thus enabling future studies of the various Nav1.2 channel conformations involved in the gating mechanism. Structural analysis of β-PMTX may also provide valuable insight for the development of future pharmaceutical agents. β-PMTX was synthesized and structures were elucidated using solution state 2D homonuclear NMR. Interestingly, β-PMTX appears to be unfolded in an aqueous environment, but in the presence of a membrane mimetic and a membrane mimetic containing a Nav1.2 fragment, β-PMTX adopted two distinct 3D conformations. Characterization and elucidation of β-PMTX structure were performed in both detergent micelles and lipid bicelles, and interacting residues were identified.

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.

NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (KD > 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 13C‐edited/13C‐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 13C‐edited/13C‐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 13C‐edited/13C‐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.

Voltage-gated sodium channels (VGSCs) are membrane proteins that serve important functions in the central and peripheral nervous systems (C/PNS) and cardiac and skeletal muscle. Diseases that can be caused by malfunctioning VGSCs (often due to modified gating properties) include epilepsy, chronic pain, and several ailments afflicting the heart. Interestingly, peptide toxins from venomous and poisonous animals have been known to target VGSCs. Site 3 and 4 peptide neurotoxins are known to interact with the voltage-sensing domains of VGSCs, modifying their gating properties. It has been proposed that peptide neurotoxins may be used directly or as lead compounds for rational drug design to alleviate symptoms caused by channelopathies. An example of this approach is the drug Prialt, an ω-conotoxin, used to alleviate intractable pain in patients. By probing the structural details of the interaction between peptide neurotoxins and VGSCs, we will gain more insight into the function of these toxins and what governs their strong and specific effect. VGSCs are large, highly hydrophobic and heavily post-translationally modified proteins, thus making it unlikely that X-ray crystallography, solution-state NMR or cryo-EM will be successful in producing structural information on intact protein at the atomic level. As an alternative strategy, we are studying the main binding sequence for site 3 and 4 toxins, the S3b-S4 paddle motif, in complex with an interacting gating modifier toxin of the VGSC NaV1.5. VSD IV of NaV1.5 is the known target of many site-3 α-scorpion and sea anemone toxins that inhibit fast inactivation of the channel. We have chemically and biosynthetically synthesized the 37 residue paddle motif peptide and characterized it through circular dichroism spectroscopy, MALDI-TOF mass spectrometry, and solution state NMR, producing the first backbone assignments of a mammalian VGSC paddle motif.

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.

The peptide T20, which corresponds to a sequence in the C‐terminal segment of the HIV ‐1 transmembrane glycoprotein gp41, is a strong entry inhibitor of HIV ‐1. It has been assumed that T20 inhibits HIV ‐1 infection by binding to the trimer formed by the N‐terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N‐ and C‐terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism of HIV ‐1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/ CD 4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC 4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. The present study concludes that an amphiphilic helix on the T20 C‐terminus binds through mostly hydrophobic interactions to a nonpolar gp120 surface formed primarily by the C4 region. The ten‐ to thousand‐fold difference between the EC 50 of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV ‐1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti‐ HIV therapeutics.

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.

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.

The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS.

ABSTRACTStructural analysis by NMR of G protein‐coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine‐activating reagents: 5‐5′‐dithiobis(2‐nitrobenzoic acid) [DTNB], 2,2′‐dithiobis(5‐nitropyridine) [DTNP], and 4,4′‐dipyridyl disulfide [4‐PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N‐terminal (NT) and C‐terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha‐factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4‐PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102‐residue fragment, 2TM‐tail [Ste2p(G31‐I120C)], was heterodimerized with CT‐EL1‐tailDTNP at pH 4.6 with a yield of ∼75%. A 132‐residue fragment, 2TMlong‐tail [Ste2p(M1‐I120C)], was expressed in both unlabeled and 15N‐labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180‐residue segmentally labeled 3TM protein that was found to be segmentally labeled using [15N,1H]‐HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 16–29, 2014.

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell‐surface receptors belonging to the G‐protein‐coupled receptor (GPCR) superfamily. The interactions between N–terminal (Nt‐) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using 1H‐15N‐HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt‐CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt‐CCR5 binding resembles the positively charged surface in HIV‐1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of β2–β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.