High-resolution NMR structure of the chemically-synthesized melanocortin receptor binding domain AGRP(87-132) of the agouti-related protein.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel beta sheet, where the last two strands form a beta hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.

NMR structure of a minimized human agouti related protein prepared by total chemical synthesis.

The structure of the chemically synthesized C-terminal region of the human agouti related protein (AGRP) was determined by 2D 1H NMR. Referred to as minimized agouti related protein, MARP is a 46 residue polypeptide containing 10 Cys residues involved in five disulfide bonds that retains the biological activity of full length AGRP. AGRP is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRP's exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 (MC3r, MC4r), and MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C-terminal regions are approximately 40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C-terminal regions. The NMR structure of MARP reported here can be characterized as three major loops, with four of the five disulfide bridges at the base of the structure. Though its fold is well defined, no canonical secondary structure is identified. While previously reported structural models of the C-terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, we find that Cys spacing is not sufficient to correctly determine the 3D fold of the molecule.

An NMR investigation of the conformational effect of nitroxide spin labels on Ala-rich helical peptides.

Nitroxide spin labels, in conjunction with electron spin resonance (ESR) experiments, are extensively employed to probe the structure and dynamics of biomolecules. One of the most ubiquitous spin labeling reagents is the methanethiosulfonate spin label which attaches a spin label selectively to Cys residues via a disulfide bond (Cys-SL). However, the actual effect of the nitroxide spin label upon the conformation of the peptide or protein cannot be unambiguously determined by ESR. In this study, a series of 16-residue Ala-rich helical peptides was characterized by nuclear magnetic resonance techniques. The C alpha H chemical shift analysis, NOEs, and 3JNH alpha coupling constants for peptides with no Cys, free Cys, and Cys-SL (with the N-O group reduced) were compared. These results indicate that while replacement of an Ala with a Cys residue causes a loss of overall helical structure, the Cys-SL residue is helix supporting, as would be expected for a non-beta-branched aliphatic amino acid. Thus, the Cys-SL residue does not perturb helical structure and, instead, exhibits helix-stabilizing characteristics similar to that found for Ala, Met, and Leu.

Estimating the relative populations of 3(10)-helix and alpha-helix in Ala-rich peptides: a hydrogen exchange and high field NMR study.

Recent experimental and theoretical work suggests that alanine-rich peptides fold as a mixture of 3(10)-helix (i --> i + 3 hydrogen bonding) and alpha-helix (i --> i + 4 hydrogen bonding). In order to assess the relative proportions of the two conformers, NMR studies were performed on the 16 residue sequences: Ac-AAAAKAAAAKAAAAKA-NH2 (3K) and Ac-AMAAKAWAAKAAAARA-NH2 (MW). Hydrogen/deuterium-exchange kinetics measured for the first three amide protons of the 3K peptide indicate that the NH of Ala3 is partially protected from exchange. This result is consistent with the presence of an i --> i + 3 hydrogen bond between the carbonyl group of the acetyl blocking group and the NH group of Ala3. The MW peptide is a modified version of the 3K peptide, designed to increase alphaH signal dispersion. 1H NMR spectra of the MW peptide at 750 MHz reveal a series of intermediate range (NOEs) consistent with a mixture of 3(10)-helix and alpha-helix. The relative intensities of the alphaN(i,i + 3) and alphabeta(i,i + 3) (nuclear Overhauser enhancements) NOEs suggest that 3(10)-helix is present throughout the peptide, but with the greatest contribution at the termini. A model was developed to determine the relative contributions of 3(10)-helix and alpha-helix. Lower bounds for the population of 3(10)-helix are approximately 50% at the termini and 25% in the middle of the peptide. The greatest alpha-helical content is between the middle of the peptide and the N terminus.

Insight into a random coil conformation and an isolated helix: structural and dynamical characterisation of the C-helix peptide from hen lysozyme.

A 17 residue peptide corresponding to the C-helix of hen lysozyme (residues 86 to 102) has been investigated in detail to assess the factors that determine its conformation in both aqueous and trifluoroethanol (TFE) solutions. A thorough characterisation of the peptide by CD and NMR techniques under both conditions has been performed including the determination of complete NMR proton sequential assignments, and measurement of NOE effects, 3JHN alpha coupling constants, temperature coefficients and residue-specific hydrogen-exchange rates. In water, the peptide adopts a largely unstructured conformation and NMR data, particularly coupling constants and chemical shift deviations, have been shown to agree closely with predictions from a model for a random coil based on the phi, psi distributions in a protein database. This indicates that under these conditions the intrinsic conformational preferences of the individual amino acid residues are the dominating factors that determine the population of conformers adopted. With increasing concentrations of TFE a cooperative transition to an extensively helical conformation occurs and the resultant changes in C alpha H chemical shifts have been shown to correlate with the changes in phi, psi populations. Using NOE and coupling constant data for this state, an ensemble of structures has been calculated and provides a model for a helix in the absence of tertiary interactions. In this model fluctuations, which increase in amplitude towards the termini, occur about the average helical phi, psi angles and are responsible for increasing the values of 3JHN alpha coupling constants above those anticipated for a static helix. The residue-specific rates of hydrogen exchange for the peptide in 50% TFE-d, are consistent with such a model, the maximum protection from exchange being observed for residues in the centre of the helix.

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHN alpha coupling constants.

Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar C alpha H-NH couplings (3JHN alpha) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the C alpha H region of 1H 2D NMR spectra. Quantitative determination of all possible 3JHN alpha values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson. 99, 552-560] have recently developed a method for determining 3JHN alpha from NOESY spectra. Because 3JHN alpha may be determined from 2D peaks outside of the C alpha H region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that 3JHN alpha can be obtained for every residue in the helical peptide Ac-(AAAAK)3A-NH2. The resulting 3JHN alpha profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.

Analysis of main chain torsion angles in proteins: prediction of NMR coupling constants for native and random coil conformations.

Using a data base of 85 high resolution protein crystal structures the distributions of main chain torsion angles, both in secondary structure and in coil regions where no secondary structure is present, have been analysed. These torsion angle distributions have been used to predict NMR homonuclear and heteronuclear coupling constants for residues in secondary structure using known Karplus relationships. For alpha helices, 3(10) helices and beta strands mean predicted 3JHN alpha coupling constants are 4.8, 5.6 and 8.5 Hz, respectively. These values differ significantly from those expected for the ideal phi angles (3.9, 3.0 and 8.9 Hz; phi = -57 degrees, -49 degrees, -139 degrees for alpha and 3(10) helices and beta strands (antiparallel), respectively) in regular secondary structure, but agree well with available experimental NMR data for nine proteins. The crystallographic data set has also been used to provide a basis for interpreting coupling constants measured for peptides and denatured proteins. Using a model for a random coil, in which all residues adopt distributions of phi, psi angles equivalent to those seen for residues in the coil regions of native folded proteins, predicted 3JHN alpha values for different residue types have been found to range from 5.9 Hz and 6.1 Hz for glycine and alanine, respectively, to 7.7 Hz for valine. A good correlation has been found between the predicted 3JHN alpha coupling constants for this model and experimental values for a set of peptides that other evidence suggest are highly unstructured. For other peptides, however, deviations from the predictions of the model are clear and provide evidence for additional interactions within otherwise disordered states. The values of homonuclear and heteronuclear coupling constants derived from the protein data base listed here therefore provide a basis not only for analysing the secondary structure of native proteins in solution but for assessing and interpreting the extent of structure present in peptides and non-native states of proteins.

Native-like secondary structure in a peptide from the alpha-domain of hen lysozyme.

BACKGROUND: To gain insight into the local and nonlocal interactions that contribute to the stability of hen lysozyme, we have synthesized two peptides that together comprise the entire alpha-domain of the protein. One peptide (peptide 1-40) corresponds to the sequence that forms two alpha-helices, a loop region, and a small beta-sheet in the N-terminal region of the native protein. The other (peptide 84-129) makes up the C-terminal part of the alpha-domain and encompasses two alpha-helices and a 3(10) helix in the native protein. RESULTS: As judged by CD and a range of NMR parameters, peptide 1-40 has little secondary structure in aqueous solution and only a small number of local hydrophobic interactions, largely in the loop region. Peptide 84-129, by contrast, contains significant helical structure and is partially hydrophobically collapsed. More specifically, the region corresponding to helix C in native lysozyme is disordered, whereas regions corresponding to the D and 3(10) helices in the native protein are helical in this peptide. The structure in peptide 84-129 is at least partly stabilized by interactions between residues in the two helical regions, as suggested by further NMR analysis of three short peptides corresponding to the individual helices in this region of the native protein. CONCLUSIONS: Stabilization of structure in the sequence 1-40 appears to be facilitated predominantly by long-range interactions between this region and the sequence 84-129. In native lysozyme, the existence of two disulphide bonds between the N- and C-terminal halves of the alpha-domain is likely to be a major factor in their stabilization. The data show, however, that native-like secondary structure can be generated in the C-terminal portion of the alpha-domain by nonspecific and nonnative interactions within a partially collapsed state.