The antiparallel pancreatic polypeptide fold in the binding of neuropeptide Y to Y1 and Y2 receptors.

Neuropeptide Y (NPY) belongs to the pancreatic polypeptide fold (PP-fold) family of regulatory peptides. Analysis of circular dicroic spectra of NPY showed that it has a high degree of secondary structure in aqueous solution which is in agreement with the globular, folded crystal structure of PP. Using three different approaches with synthetic peptides, we have probed the importance of the PP-fold structure in the interaction of NPY with two types of binding sites, Y1 and Y2 receptors. First, stepwise construction of the NPY molecule from the C-terminal amidated end, showed that although C-terminal fragments encompassing most of the long alpha-helix reacted reasonably well with the Y2 receptor, both Y1 and Y2 receptors required the presence of both ends of the PP-fold for full activity. Second, perturbation of the PP-fold by substitution with a helix-breaking proline residue, resulted in the loss of recognition of the N-terminal segment of the molecule by both types of receptors. Finally, a hybrid analog was constructed in which the essential, but by itself inactive, C-terminal segment of NPY was joined with the PP-fold motif of PP. This segment of PP is only 43% homologous to the similar motif in NPY, and most of the common residues cluster in the hydrophobic core of the fold. Nevertheless, the hybrid analog reacted with almost full potency on the Y2 receptors. It is concluded that the antiparallel PP-fold is of structural importance for the receptor binding of NPY, and that its main function is to present the combined C- and N-terminal segments of the molecule to the receptors.

Second, perturbation of the PP-fold by substitution with a helix-breaking proline residue, resulted in the loss of recognition of the N-terminal segment of the molecule by both types of receptors. Finally, a hybrid analog was constructed in which the essential, but by itself inactive, C-terminal segment of NPY was joined with the PP-fold motif of PP. This segment of PP is only 43% homologous to the similar motif in NPY, and most of the common residues cluster in the hydrophobic core of the fold. Nevertheless, the hybrid analog reacted with almost full potency on the Yz receptors.
It is concluded that the antiparallel PPfold is of structural importance for the receptor binding of NPY, and that its main function is to present the combined C-and N-terminal segments of the molecule to the receptors.

Neuropeptide
Y (NPY)' is an important regulator of neuronal function with a widespread distribution.
In the central nervous system, NPY is involved mainly in the regulation of food intake, memory processing, and circadian rhythm. In the peripheral nervous system, NPY functions especially as a cotransmitter with norepinephine in the regulation of vascular * The work was supported in part by the Biotechnology Centre for Neuropeptide Research, the NOVO, the Carlsberg, and the Vissing Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
7 Recipient of a research professorship from the Danish Medical Research Council and the Weimann Foundation. To whom correspondence should be addressed: Laboratory for Molecular Endocrinology, Rigshospitalet 6321, Blegdamsvej 9, DK-2100, Copenhagen 0, Denmark.
tone; a fact which has changed the concept of the function of the sympathetic nervous system. The neuroanatomy and physiology of NPY has recently been reviewed in a symposium volume (1).
The primary structure of NPY has been preserved well during evolution (2, 3). The 36-amino acid, amidated peptide belongs to the pancreatic polypeptide fold (PP-fold) family of peptides, characterized by a common tertiary structure, the so-called PP-fold (4, 5). In addition to NPY, the pancreatic hormone pancreatic polypeptide (PP) and the intestinal hormone peptide YY (PYY) belong to the family (6,7). The PPfold structure of avian PP has in great detail been studied by Blundell and coworkers (8,9) by x-ray diffraction analysis down to 0.98 A resolution. The PP-fold consists of a polyproline-like helix and an amphiphilic a-helix which lies antiparallel with an angle of 152 o between the helix axes (8). The two helices are joined by a type I P-turn and are held in the folded configuration through hydrophobic interactions between side chains of the a-helix interdigitating with the prolines in the N-terminal section (Fig. 1). Circular dichroism studies of PP-fold peptides, including NPY, indicate that although the peptides do not have any stabilizing disulfide bridges, they do hold a considerable amount of secondary structure in aqueous solution (4,(9)(10)(11)(12)(13). This is true even for the monomeric form of NPY (13). As evaluated by Dobson and coworkers by two-dimensional NMR the solution structure of at least one member of the family, bovine PP, appears to be very similar to the crystal structure of avian PP (14).
In the present investigation we have probed the importance of the PP-fold motif of NPY for the binding of this peptide to two types of NPY receptors (15-17). Three different approaches were employed: 1) The structure of the NPY molecule was built up stepwise from the essential C-terminal amidated end. 2) The PP-fold was perturbed through a single substitution.
3) The PP-fold motif of NPY was exchanged with the vastly different but structurally homologous PP-fold of PP itself. The schematic structure of NPY shown at the top has been modeled over the x-ray structure of avian PP (4). The secondary structural elements are identified above the sequences. The sequence of human pancreatic polypeptide is shown at the bottom for comparison. The residues which differs from those of NPY are indicated in white on black. cedure (2 mmol of amino acid/O.2 mmol of resin) and the fragments of NPY were obtained by taking out samples of the resin at appropriate times during the synthesis (indicated in Fig. 1). The coupling was tested after each step by the quantitative ninhydrin assay (18,19). Side chain protecting groups were as follows: 2-bromobenzyloxycarbonyl for tyrosine, 4-toluenesulfonyl for arginine, 2-chlorobenzyloxycarbonyl for lysine, benzyloxymethyl for histidine, benzyl ester for aspartic and glutamic acid, except for aspartic acid residue 6, which was cyclohexyl ester. The full length peptides were cleaved from the resin by hydrogen fluoride as described (20), and the fragments were cleaved by the trifluoromethylsulfonic acid method (21). Purification-The crude peptides were extracted into 50% acetic acid after cleavage, diluted 5 times with water, and gel-filtered on a Sephadex G-25 superfine column. The column was eluted with 2% acetic acid containing 15% isopropanol. The peptide containing fractions were freeze-dried and purified by reversed phase HPLC, on a Cla column (1.6 X 20 cm, 7 Nrn particles) which was eluted at a flow of 6 ml/min with a gradient of acetonitrile from 30 to 45% in 0.2 M ammonium acetate, pH 3.5, over 45 min. Amino acid analysis, which was performed on the hydrolyzed peptides (6 M HCl, with 0.1% phenol for 18 h at 110 "C) by ion exchange chromatography on a Kontron amino acid analyzer using the lithium citrate buffer system, gave results which were in good agreement with the theoretically expected values (Table I). The amino acid sequence of NPY, NPY-(g-36), [ProzO]NPY, and [Ile31,Gln34]NPY were controled by gasphase sequencing on an Applied Biosystems model 470A Sequencer. The integrity of the C-terminal amide function was monitored by a radioimmunoassay, which is totally specific for this modification in NPY, using NPY antibody 8999 and ['251-Tyr']monoiodo-NPY (22). NPY-(13-36)-peptide was synthesized by Ferring and was a generous gift from Dr. Rolf HQkanson, University of Lund, Sweden.

Circular Dichroism
Spectroscopy Circular dichroism (CD) spectra were recorded on a Jobin Yvon Dichrograph Mark V. The CD signal was calibrated with (+)-lOcamphorsulfonic acid in a l-cm cell assuming a AC of 2.36 M-' cm-' at 290.5 nm. A spectral band width of 2.0 nm and a scan rate of 4 nm/min were used. The peptide samples were contained between cylindrical quartz windows with a nominal path length of 0.020 cm, dissolved to a final concentration of 0.1 mM in 10 mM acetic acid, pH 4.5, except for NPY-(8-36)-peptide which was dissolved in distilled water at pH 5.9. Peptide concentrations were determined by amino acid analysis and all CD measurements were made at 26 +2 "C. The final spectra were averaged from three scans prior to base-line subtraction and smoothing. The unit of Ac was based on the molar concentration of peptide bonds. The CD experiments were all performed at concentrations where NPY most likely is in a dimeric form (13). The secondary structure of the peptides was calculated from their spectrum using the methods of singular value decomposition and variable selection as described by . We chose the calfskin collagen spectrum (26) as a reference spectrum for the polyproline II-like helix structure because this polymer resembles the sequence of the NPY polyproline-like segment more than for example poly(L-proline) II does. Analysis of difference CD spectra of closely related proteins is a very sensitive method for studies on changes in secondary structure*; in the present study the difference spectrum between the experimental spectra of NPY and NPY-(8-36)-peptide and between NPY and [Pro2"]NPY were calculated.

Binding Experiments
In the present study, the SK-N-MC cell line and its subline MC-IXC were used as a general Y, receptor model and porcine hippocampal membranes were used as a general Y* receptor preparation (16, 17).
Binding to Cells-The human primitive neuroectodermal cell line SK-N-MC (and the MCIX-C subclone of this) were kindly provided by Drs. June Biedler and Barbara A. Spengler, Sloan-Kettering Memorial Institute, New York (27, 28). All media and materials for tissue culture were from Gibco. As described in detail previously (15,16), binding studies with cells were performed with 50,000 cpm of radioligand at 37 "C using triplicates of 1.2 x lo6 cells which had been preincubated for 2 days in Petri wells, 6-well culture plates (Costar), precoated with poly-Lys-Ala(Sigma).
The incubation time was 60 min.

RESULTS
CD Analysis of NPY-The CD spectrum of NPY and that of the fragment, NPY-(8-36)-peptide, which was specifically designed to lack the segment corresponding to the polyproline-like helix, are shown in Fig. 2A. The weighted difference spectrum between the experimental spectra of NPY and that of NPY-(8-36)-peptide, was compared to the spectrum of collagen (Fig. ZB). The similarity of both the shape and the intensity of these two spectra indicates that the f-7 segment of NPY does adopt a polyproline-like structure. Computational analysis of the NPY spectrum was performed both after subtraction of one-fifth of the spectrum for collagen, corresponding to the 7 out of the 35 peptide bonds which according to the crystal structure of avian PP could be expected to be in a polyproline-like helix, and after subtraction of the difference spectrum between NPY and NPY-(8-36)-peptide.
As shown in Table II, these analyses gave a-helical contents of 32 and 37%, respectively, which are only slightly less than the 43% found in the crystal structure of the homologous avian PP (4). The difference spectrum between NPY and NPY-(8-36)-peptide gave as a reference spectrum computational results for the structure of NPY which most closely resemble those of the PP crystal structure. This was most evident in the estimation of the amount of P-turn structure in NPY (Table I). In conclusion, the CD analysis supports the notion that NPY in aqueous solution holds a tertiary structure with a PP-fold similar to that found in the crystal structure of avian PP.
Binding of C-terminal Fragments of NPY-The C-terminal amide group is essential for the biological function of PP-fold peptides (30-32), and the free acid form of NPY was in our receptor preparations, as expected, devoid of any significant binding activity (data not shown). Thus, we decided to build the NPY molecule up stepwise from the C terminus. We chose to add sequences corresponding to turn after turn of the amphipathic a-helix to the C-terminal, amidated hexapeptide (Fig. 1). In the last two fragments, sequences were added which corresponded to the P-turn, and finally half of the polyproline helix. On the Yz receptors the C-terminal hexapeptide itself did not displace the radiolabeled NPY. However, NPY-(23-36)peptide and larger fragments bound to the Y, receptors (Fig.  3). NPY-(19-36)-peptide displaced radiolabeled NPY from Ya receptors with an I&, of 5.2 nM as compared to 0.74 nM for NPY. Further addition of the last turn of the helix, the fiturn, and half of the polyproline helix apparently did not increase the binding affinity of the fragments to the Ye receptors. On the Y, receptors, not even the longest fragment, NPY-(4-36)-peptide, bound significantly (Fig. 3). Thus, although the long C-terminal fragments of NPY encompassing most of the amphipathic a-helix bind rather well to the Yp receptor, both of the receptors requires the presence of even the far N-terminal segment of the molecule for full activity. Structure and Binding of NPY with Perturbed PP-fold-We decided to perform a single substitution of the tyrosine residue in position 20 with the helix-breaking imino acid, proline, to try to hinder as discretely as possible the formation of the a-helix and thus the PP-fold. The CD spectrum of [ProzO]NPY indicated that the solution structure of the analog is vastly different from that of NPY, as shown in Fig. 4. Since it was unknown whether the polyproline-like helix would be present or not in the structure of [Pro*']NPY, the calculations of secondary structural elements were performed for both cases, (Table II). In both analysis, a substantial reduction in a-helix content was found, from 32 or 37% in NPY to either 20 or 15%, dependent on the mode of calculation. This result was confirmed by analysis of the difference spectrum obtained from the experimental spectra of NPY and [Pro"]NPY (data not shown). Thus, the substitution had the desired deleterious effect on the structure of the NPY molecule.
In Yp receptor preparations, the analog, [Pro2']NPY, displaced radiolabeled NPY with an ICso of 5.6 nM similar to that of NPY-(19-36)-peptide (Fig. 5). [Pro"]NPY did not bind at all to Y1 receptors (Fig. 5). Thus, the analog in which the secondary structure in aqueous solution had been seriously disturbed reacted with both Y1 and Yz receptors as the "corresponding" C-terminal fragment, NPY-(19-36)-peptide. The structural information, which is present in the intact Nterminal segment of the unfolded analog, apparently cannot be recognized by the receptors.
Binding of NPY with a Functional but Altered PP-fold-The C-terminal end of NPY is essential for the receptor binding but the C-terminal hexapeptide is by itself without potency (Fig. 3) In A is shown the spectra of NPY (-) and NPY-(a-36)peptide (-----), dissolved in 10 mM acetic acid and distilled water, respectively, to final concentrations of 0.13 IUM NPY and 0.15 mM fragment, and to pH 4.5 and 5.9, respectively. B shows the difference spectrum ( -) calculated as a weighted difference between the experimental spectra for NPY and NPY-(S-36)-peptide shown in A, together with the CD spectrum of calfskin collagen (----), redrawn from Ref. 26. Both spectra have been reduced in intensity by a factor of 0.20 to make them comparable to the spectra shown in A.

hexapeptide
of NPY with the PP-fold of human PP and thereby create a hybrid molecule with the correct C terminus of NPY but a vastly different PP-fold motif (Fig. 1). Although the hybrid, PP-(l-30)-NPY-(31-36)-hybrid, had 17-amino acid substitution as compared to NPY it reacted with almost full potency on the Yp receptors (Fig. 6). The hybrid did, however, not bind to Y1 receptors (Fig. 6). This is in accordance with the fact that the N-terminal sequence of the analog differs from that of NPY, and this part of NPY is essential for its binding to Y, type of receptors, as shown in Fig. 3, lower panel.

DISCUSSION
In the present study we provide evidence for the importance of the secondary and tertiary structure of NPY for its biological function. Kaiser and Kezdy (33) have made an ample case for the general importance of secondary structural elements in the binding and function of medium size peptides, 25-50 amino acids. They focused mainly on a series of peptides in which a spatial segregation of hydrophobic and hydrophilic residues creates a complementary amphiphilicity in a major segment of secondary structure (34). The peptides which they studied do not hold the ordered secondary structures in aqueous solution; however, the structures are believed to form in the amphiphilic environment of the cell surfaces. The formation and functional importance of especially amphiphilic helical segments have been probed in, for example, meilitin (35), calcitonin (36), and P-endorphin (37). The experimental approach was to replace a segment of the hormone with a nonhomologous model peptide which preserved the general secondary structure, e.g. an amphiphilic a-helix composed of D-amino acids. Thus, by minimizing the sequence homology, even including nonpeptide elements (38), and still reproducing the biological response, it has been possible to provide evidence for the functional importance of segments of amphiphilic secondary structure (33, 34). This "peptide engineering" approach could not directly be applied to NPY. Although peptides have been produced which had the desired physiochemically properties, these analogs bound with less than 1% of the potency of NPY to brain receptors (13). Nevertheless, the observation in the present study, that partial disruption of the central helix in NPY through the introduction of a single proline residue secludes the N-terminal sequence from receptor recognition, supports the conjecture that secondary structure is important for the function of the peptide. It should be emphasized, however, that NPY differs from endorphin, calcitonin, etc. in holding a globular, tertiary structure in aqueous solution; a structure in which the amphiphilic helix is stabilized through intramolecular, hydrophobic interactions. Thus, for the final test of the importance of the PP-fold we decided to apply the classical approach of Kaiser and K6zdy in a slightly modified form. The structural element to be probed, the PP-fold of NPY, was exchanged with the corresponding segment of the homologous peptide, pancreatic polypeptide.
In other words we used a similar peptide segment which is very different in sequence (see Fig. 1) but which has been structurally designed through the evolutionary refinement of nature, not man. The hybrid peptide bound equally well as NPY to NPY-Y2 receptors (Fig. 6), which strongly indicates that the PP-fold is an important but purely structural element in the interaction of NPY with the YZ receptor. In fact, peptide PYY (7), which also binds with full potency to NPY receptors (15,16,32), is similar to the hybrid analog, since 12 of the 31 amino acid residues in the PP-fold area differ between NPY and PYY. Among the 23 different PP-fold peptides characterized today, there is a strong preservation of the core residues, described by Glover et al. (4), which stabilizes the PP-fold structure, whereas the surface residues vary freely among hydrophilic residues (5).
The receptors for PP-fold peptides appear to recognize the combined C-terminal and N-terminal segments of the molecules brought together by the inter-exchangeable PP-fold