Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy.

The structural characterization of peptide hormones and their interaction with G-protein (guanine nucleotide-binding regulatory protein) coupled receptors by high-resolution nmr is described. The general approaches utilized can be categorized into three different classes based on their target: the ligand, the receptor, and the ligand/receptor complex. Examples of these different approaches, aimed at facilitating the rational design of peptides and peptidomimetics with improved pharmacological profiles, based on work carried out in our own laboratory, are given. In the ligand-based approach, the high-resolution structures of bradykinin analogues allowing for the development of a structure-activity relationship for activation of the B1 receptor are described. Studies targeting the receptor are to a large extent theoretical, based on computational molecular modeling. However, experimentally based structural features provided by high-resolution nmr can be used to great advantage, providing insight into the mechanism of receptor function, as illustrated here with results from parathyroid hormone. A similar combination of theoretical methods, supplemented by high-resolution structures from nmr has been utilized to probe the formation and stabilization of the ligand/receptor complex both for parathyroid hormone and cholecystokinin. In each of these three approaches, the importance of well-designed peptide mimetics and accurate structural analysis by high-resolution nmr, will be highlighted.     

Structural proteomics by NMR spectroscopy

Structural proteomics is one of the powerful research areas in the postgenomic era, elucidating structure-function relationships of uncharacterized gene products based on the 3D protein structure. It proposes biochemical and cellular functions of unannotated proteins and thereby identifies potential drug design and protein engineering targets. Recently, a number of pioneering groups in structural proteomics research have achieved proof of structural proteomic theory by predicting the 3D structures of hypothetical proteins that successfully identified the biological functions of those proteins. The pioneering groups made use of a number of techniques, including NMR spectroscopy, which has been applied successfully to structural proteomics studies over the past 10 years. In addition, advances in hardware design, data acquisition methods, sample preparation and automation of data analysis have been developed and successfully applied to high-throughput structure determination techniques. These efforts ensure that NMR spectroscopy will become an important methodology for performing structural proteomics research on a genomic scale. NMR-based structural proteomics together with x-ray crystallography will provide a comprehensive structural database to predict the basic biological functions of hypothetical proteins identified by the genome projects.     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy

Four betacyanin pigments were analysed by LC NMR and subjected to extensive NMR characterisation after isolation. Previously, low pH values were applied for NMR investigations of betalains resulting in rapid degradation of the purified substances thus preventing extensive NMR studies. Consequently, up to now only one single (13)C NMR spectrum of a betalain pigment, namely that of neobetanin (=14,15-dehydrobetanin), was available. Because of its sufficient stability under highly acidic conditions otherwise detrimental for betacyanins, this pigment remained an exemption. Since betalains are most stable in the pH range of 5-7, a new solvent system has been developed allowing improved data acquisition through improved pigment stability at near neutral pH. Thus, not only (1)H, but for the first time also partial (13)C data of betanin, isobetanin, phyllocactin and hylocerenin isolated from red-purple pitaya [Hylocereus polyrhizus (Weber) Britton & Rose, Cactaceae] could be indirectly obtained by gHSQC- and gHMQC-NMR experiments.     

The conformation of porcine-brain natriuretic peptide by two-dimensional NMR spectroscopy

Two-dimensional (2D) 1H-NMR spectra of porcine-brain natriuretic peptide (pBNP) have been recorded at 300 MHz and 400 MHz. Peak assignments have been made and the combined information from chemical shifts, coupling constants, temperature coefficients and NOEs have been used to determine the conformational properties of pBNP in (C2H3)2SO. Overall the peptide appears to be flexible, with the possibility of some beta-type structure near the C terminus. Some of the assignments and deduced structural features in the current study differ from those in a recent report by Inooka et al. [Inooka, H., Kikuchi, T., Endo, S., Ishibashi, Y., Wakimasu, M. and Mizuta, E. (1990) Eur. J. Biochem. 193, 127-134] which may indicate the sensitivity of the structure of this peptide to differences in solution conditions.     

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.     

Structural studies of porcine myeloid antibacterial peptide PMAP-23 and its analogues in DPC micelles by NMR spectroscopy.

PMAP-23 is a cathelicidin-derived antimicrobial peptide identified from porcine leukocytes. PMAP-23 was reported to show potent antimicrobial activity against Gram-negative and Gram-positive bacteria without hemolytic activity. To study the structure-antibiotic activity relationships of PMAP-23, two analogues by replacing Trp with Ala were synthesized and their tertiary structures bound to DPC micelles have been studied by NMR spectroscopy. PMAP-23 has two alpha-helices, one from Arg1 to Arg10 in the N-terminal region and the other from Phe18 to Arg23 in the C-terminal region. PMAP-1 (Trp(7)-->Ala) shows similar structure to PMAP-23, while PMAP-2 (Trp(21)-->Ala) has a random structure in the C-terminus. PMAP-2 was found to show less antibacterial and vesicle-disrupting activities than PMAP-23 and PMAP-1 [J. H. Kang, S. Y. Shin, S. Y. Jang, K. L. Kim, and K.-S. Hahm (1999) Biochem. Biophys. Res. Commun. 264, 281-286]. Trp(21) in PMAP-23 which induces an alpha-helical structure in the second alpha-helix is essential for the antibacterial activity of PMAP-23. Also, the fluorescence data proved that Trp(21) at the second alpha-helix is buried deep into the phospholipid in the membrane. Therefore, it implies that Trp(21) in the second alpha-helix at the C-terminus of PMAP-23 may play an important role on the interactions with the membrane and the flexible region including two proline residues may allow this alpha-helix to span the lipid bilayer.     

Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy

A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein.     

Structural analysis of phenolic glucosides from salicaceae by NMR spectroscopy

Some characteristic features of the NMR spectra of phenolic glycosides are described in order to facilitate the structural determination of these compounds.     

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

The bioactive conformation of glucose-dependent insulinotropic polypeptide by NMR and CD spectroscopy

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal incretin hormone, which modulates physiological insulin secretion. Because of its glucose-sensitive insulinotropic activity, there has been a considerable interest in utilizing the hormone as a potential treatment for type 2 diabetes. Structural parameters obtained from NMR spectroscopy combined with molecular modeling techniques play a vital role in the design of new therapeutic drugs. Therefore, to understand the structural requirements for the biological activity of GIP, the solution structure of GIP was investigated by circular dichroism (CD) followed by proton nuclear magnetic resonance (NMR) spectroscopy. CD studies showed an increase in the helical character of the peptide with increasing concentration of trifluoroethanol (TFE) up to 50%. Therefore, the solution structure of GIP in 50% TFE was determined. It was found that there was an alpha-helix between residues 6 and 29, which tends to extend further up to residue 36. The implications of the C-terminal extended helical segment in the inhibitory properties of GIP on gastric acid secretion are discussed. It is shown that the adoption by GIP of an alpha-helical secondary structure is a requirement for its biological activity. Knowledge of the solution structure of GIP will help in the understanding of how the peptide interacts with its receptor and aids in the design of new therapeutic agents useful for the treatment of diabetes.     

Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and methylation analysis

Two galactofuranomannans, Ths-4 and Ths-5, were isolated from the lichen, Thamnolia vermicularis var. subuliformis, using ethanol fractionation and anion-exchange and size-exclusion chromatography. The average molecular weights of Ths-4 and Ths-5 were estimated to be 19 and 200 kDa, respectively. Structural characterisation of Ths-4, Ths-5 and their partially hydrolysed derivatives was performed by methanolysis and methylation analysis. The intact and partially hydrolysed Ths-4 was further analysed using NMR spectroscopy (1D, COSY, NOESY, TOCSY, HSQC and HMBC). According to the data obtained, the heteroglycans Ths-4 and Ths-5 have similar structures, but have large differences in molecular weight. The structure is composed of 3-O-linked and 5-O-linked galactofuranosyl chains linked to a mannan core. The mannan core consists of a main chain of alpha-(1-->6)-linked mannopyranosyl residues, substituted at O-2 with either a single alpha-mannopyranosyl unit or an alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown.     

Structural characterization of the N-terminal kinase-interacting domain of an Hsp90-cochaperone Cdc37 by CD and solution NMR spectroscopy

Cdc37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between Cdc37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in Cdc37 molecule, little is known about the structure of the N-terminal domain of Cdc37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of Cdc37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of Cdc37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of Cdc37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of Cdc37 with various kinases.     

Structural studies of wheat flour glutenin polymers by CD spectroscopy

A dissolution procedure of unreduced glutenin polymers of three wheat flour varieties (WRU 6981, Alisei 1, and Alisei 2) by sonication in the presence of SDS (sodium dodecyl sulphate), after the elimination of albumins, globulins, and gliadins, was achieved, and the molecular weight distribution of glutenin polymers obtained by this method was measured by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. A structural study by CD spectroscopy at different temperatures of WRU 6981 glutenin polymer and of 1Ax1 high-M(r) (relative molecular mass) glutenin subunit, which is the only high-M(r) subunit contained in WRU 6981 flour, was undertaken to understand if the information obtained from the single subunit were applicable to the total polymer. CD spectroscopy also has been employed to study the glutenin polymers obtained by Alisei 1 and Alisei 2 wheat flours; Alisei 1 biotype contained 1Bx7 and 1Dx2+1Dy12 high-M(r) subunits, whereas the Alisei 2 biotype contained only 1Bx7 and 1Dy12 subunits. A conformational study was undertaken by CD spectroscopy at different temperatures and in the presence of some chemical denaturant agents, such as urea and sodium dodecyl sulphate, in order to obtain information about their intrinsic stability and to verify if the 1Dx2 subunit presence determined a different structural behavior between Alisei 1 and Alisei 2 polymers. MALDI-TOF mass spectrometric experiments showed that the glutenin polymers molecular weights were in the mass range of 500000-5000000. CD spectra indicated that a single conformational state did not predominate in the temperature range studied but equilibrium between two distinct conformational states existed; moreover, all the changes induced by urea and by SDS followed a multistep transition process.     

Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy

This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid alpha-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid alpha-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual alpha-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu(41), Leu(45), and Leu(48)) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.     

Conformational studies of RGDechi peptide by natural‐abundance NMR spectroscopy

Integrins are heterodimeric cell‐surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin‐inhibitor drugs have been developed for integrin‐based medical applications, the structural determinants modulating integrin‐ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C‐terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for α_vβ3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of α_vβ₃ by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of α_vβ₃ integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance ¹⁵N and ¹³C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.     

Structural determination of the vasoactive intestinal peptide by two-dimensional H-NMR spectroscopy

The structure of the vasoactive intestinal peptide 1-28 in 40% 2,2,2-trifluoroethanol was investigated by two-dimensional 1H-nmr spectroscopy. All 1H resonances, except the gamma, delta, and epsilon protons of the lysine residues, could be sequentially assigned. Numerous intraresidual as well as short-range interresidual nuclear Overhauser effect spectroscopy connectivities were observed. Using a variable-target function minimization, a molecular model consisting of two helical stretches involving residues 7-15 and 19-27 connected by a region of undefined structure was calculated. The existence of an undefined structure between residues 16 and 18 confers mobility to the peptide molecule.