Search

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil–helix transitions could be the basis of polyamine regulation in contemporary proteins.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent‐driven exchanges to enhance NOE correlations between exchangeable and non‐exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross‐peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS‐CoV‐2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts., An approach to enhance the sensitivity of 2D NOESY correlations among labile protons is introduced and exemplified with experiments on RNA fragments derived from the SARS‐CoV‐2 genome and with other samples. The approach leads to experiments that are ca. 10‐fold faster and 3‐fold more sensitive than conventional counterparts. The mechanism of this sensitivity boost is explained, and its strengths and limitations are analyzed.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remains unclear. Further, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein to glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyper-acidic protein at sub-millimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+and Ca2+had an effect similar to a di-amine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues, and that (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Previous studies conducted on flexible loop regions in proteins revealed that the energetic consequences of changing loop length predominantly arise from the entropic cost of ordering a loop during folding. However, in an earlier study of human acylphosphatase (hmAcP) using experimental and computational approaches, we showed that thermodynamic stabilization upon loop truncation can be attributed mainly to the increased entropy of the folded state. Here, using 15N NMR spectroscopy, we studied the effect of loop truncation on hmAcP backbone dynamics on the picosecond-nanosecond timescale with the aim of confirming the effect of folded state entropy on protein stability. NMR-relaxation-derived N-H squared generalized order parameters reveal that loop truncation results in a significant increase in protein conformational flexibility. Comparison of these results with previously acquired all-atom molecular dynamics simulation, analyzed here in terms of squared generalized NMR order parameters, demonstrates general agreement between the two methods. The NMR study not only provides direct evidence for the enhanced conformational entropy of the folded state of hmAcP upon loop truncation but also gives a quantitative measure of the observed effects.

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.The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505.

NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-α2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.

Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) alpha-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by alpha-bungarotoxin (alpha-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and alpha-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to alpha-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of alpha-BTX to the AChR with an IC(50) of 2 nM. The solution and crystal structures of complexes of alpha-BTX with HAP were solved, demonstrating that the HAP fits snugly to alpha-BTX and adopts a beta-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that alpha-BTX wraps around the receptor binding-site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand-binding site. Our proposed model explains the strong antagonistic activity of alpha-BTX and accommodates much of the biochemical data on the mode of interaction of alpha-BTX with the AChR.

Two-dimensional nuclear magnetic resonance (2D NMR) provides one of the foremost contemporary tools available for the elucidation of molecular structure, function, and dynamics. Execution of a 2D NMR experiment generally involves scanning a series of time-domain signals S(t(2)), as a function of a t(1) time variable which undergoes parametric incrementation throughout independent experiments. Very recently, we proposed and demonstrated a general approach whereby this serial mode of data acquisition is parallelized, enabling the acquisition of complete bidimensional NMR data sets via the recording of a single transient. The present paper discusses in more detail various conceptual and experimental aspects of this novel 2D NMR methodology. The basic principles of the approach are reviewed, various homo- and heteronuclear NMR applications are illustrated, and the main features and artifacts affecting the method are derived. Extensions to higher-dimensional experiments are also briefly noted.

A scheme enabling the complete sampling of multidimensional NMR domains within a single continuous acquisition is introduced and exemplified. Provided that an analyte's signal is sufficiently strong, the acquisition time of multidimensional NMR experiments can thus be shortened by orders of magnitude. This could enable the characterization of transient events such as proteins folding, 2D NMR experiments on samples being chromatographed, bring the duration of higher dimensional experiments (e.g., 4D NMR) into the lifetime of most proteins under physiological conditions, and facilitate the incorporation of spectroscopic 2D sequences into in vivo imaging investigations. The protocol is compatible with existing multidimensional pulse sequences and can be implemented by using conventional hardware; its performance is exemplified here with a variety of homonuclear 2D NMR acquisitions.

a-Bungarotoxin (a-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind a-BTX and inhibit its binding to AChR. Structure- based design concomitant with biological information of the a-BTXyAChR system yielded 13-mer peptides that bind to a-BTX with high affinity and are potent inhibitors of a-BTX binding to AChR (IC of 2 nM). X-Ray and NMR 50 spectroscopy reveal that the high-affinity peptides fold into an anti-parallel b-hairpin structure when bound to a- BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule. 2002 Elsevier Science B.V. All rights reserved.

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.

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.

The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to alpha-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.

Background: α-Bungarotoxin (α-BTX) is a highly toxic snake venom α-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind α-BTX with high affinity, and inhibit its interaction with AChR with an IC 50 of 2 nM. The design of these peptides was based on a lead peptide with an IC 50 of 3×10 −7 M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. Results: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of α-BTX with the lead peptide, as well as structure–function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC 50 values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against α-BTX lethality. Conclusions: Synthetic peptides with high affinity to α-BTX may be used as potential lead compounds for developing effective antidotes against α-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

The exopolysaccharide of Bacillus licheniformis ATCC 9945 (formerly B. subtilis ATCC 9945) contains among other glycoses 4-acetamido-2-amino-2,4,6-trideoxy-D-glucose, termed N-acetylbacillosamine (Bac2N4NAc). A similar diamino glycose, 2-acetamido-4-amino-2,4,6-trideoxy-D-glucose, was found in a surface layer (S-layer) glycoprotein preparation of Clostridium symbiosum HB25. Electron microscopic studies, however, showed that B. licheniformis ATCC 9945 is not covered with an S-layer lattice, indicating that the N-acetylbacillosamine present in that organism might be a constituent of a cell wall-associated polymer. For elucidation of the structure of the N-acetylbacillosamine-containing polysaccharide, it was purified from a trichloroacetic acid extract of B. licheniformis ATCC 9945 cells. Using different hydrolysis protocols and a hydrolysate of the S-layer glycoprotein preparation from C. symbiosum HB25 as reference, the purified polysaccharide was found to contain 2,4-diamino-2,4,6-trideoxy-glucose, 2-acetamido-2-deoxy-glucose, 2-acetamido-2-deoxy-galactose and galactose in a molar ratio of 1 : 1 : 1 : 2. One- and two-dimensional NMR spectroscopy, including 800 MHz proton magnetic resonance measurements, in combination with chemical modification and degradation experiments, revealed that the polysaccharide consists of identical pyruvylated pentasaccharide repeating units with the structure: [-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-alpha-D-GlcpNAc-(1-->3)-beta-D-Bacp2N4NAc-(1-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-beta-D-GalpNAc-(1-->](n)

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 (1)H-(15)N-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.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.

The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The VH sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a beta-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 A2, about half of the contact area observed in protein-antibody complexes.

The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact β-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.

Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) alpha-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by alpha-bungarotoxin (alpha-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and alpha-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to alpha-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of alpha-BTX to the AChR with an IC(50) of 2 nM. The solution and crystal structures of complexes of alpha-BTX with HAP were solved, demonstrating that the HAP fits snugly to alpha-BTX and adopts a beta-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that alpha-BTX wraps around the receptor binding-site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand-binding site. Our proposed model explains the strong antagonistic activity of alpha-BTX and accommodates much of the biochemical data on the mode of interaction of alpha-BTX with the AChR.

a-Bungarotoxin (a-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind a-BTX and inhibit its binding to AChR. Structure- based design concomitant with biological information of the a-BTXyAChR system yielded 13-mer peptides that bind to a-BTX with high affinity and are potent inhibitors of a-BTX binding to AChR (IC of 2 nM). X-Ray and NMR 50 spectroscopy reveal that the high-affinity peptides fold into an anti-parallel b-hairpin structure when bound to a- BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule. 2002 Elsevier Science B.V. All rights reserved.

A spin-labeled peptide antigen (TEMPOVEVPGSQHIDSQ) was used to measure NOESY difference spectra that show interactions in the binding site region of the Fab fragment of the anti-cholera toxin peptide antibody TE33. In addition to identification of peptide-Fab interactions and interactions within the bound peptide, these difference spectra show well-resolved cross peaks due to interactions within the large Fab fragment (50 kDa). These difference spectra indicate that the conformational changes in the Fab upon peptide binding are confined to the combining site region of the antibody. The NOESY difference spectra of selectively deuterated Fab molecules were used in combination with HOHAHA measurements to assign the interactions to amino acid type and to identify the interactions within the Fab as either inter- or intraresidue interactions. The assignment of interactions within the Fab to corresponding aromatic residues in the Fab sequence was facilitated by an earlier NMR-derived model calculated on the basis of NOE restraints on Fab-peptide and intra-bound-peptide distances. The new restraints on distances within the Fab, combined with the previously obtained restraints, were used to generate a refined NMR-derived model for the TE33-peptide complex.

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody–peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab–peptide complex (Fab: antibody fragment made of Fv—the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen—the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3). which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. © 1994 John Wiley & Sons, Inc.

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of anti-peptide antibodies with native proteins it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen and the bound peptide conformation. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the information concerning antibody-peptide interactions and intramolecular interactions in the bound ligand from the crowded and unresolved spectrum of the Fab complex. These measurements yield restraints on interproton distances in the complex which are used to dock the peptide into calculated models for the antibodies' combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3), which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by antibodies, the structure of their combining sites and relationships between antibodies' primary structure, and their interactions with peptide antigens.

The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.

The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE). Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50,000.(ABSTRACT TRUNCATED AT 250 WORDS)