Bovine Pancreatic Polypeptide (bPP) Undergoes Significant Changes in Conformation and Dynamics upon Binding to DPC Micelles

doi:10.1016/S0022-2836(02)00889-6

Journal of Molecular Biology

Volume 322, Issue 5, 4 October 2002, Pages 1117-1133

https://doi.org/10.1016/S0022-2836(02)00889-6 Get rights and content

Abstract

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08 ns derived from the ¹⁵N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4–34.

The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined α-helical region comprising residues 17–31. The average RMSD value for residues 17–31 is 0.22(±0.09) Å. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4–12 and low values of the generalized order parameter S². The membrane–peptide interface was investigated by micelle-integrating spin-labels and H,²H exchange measurements. It is formed by those residues which make contacts between the C-terminal α-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y₄ receptor.

Introduction

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. These regulatory peptides include the endocrinic peptides PP, the peptide YY (PYY) and the neurotransmitter NPY. Pancreatic polypeptides are synthesized as part of a larger precursor and are localized mainly in endocrinic cells in the pancreas,¹ from which they are released into the circulation after ingestion of a meal.² In the central nervous system PP promotes food intake and gastric emptying by activation of the Y₄ and probably Y₅ receptors.3., 4.

The members of the NPY family activate a heterogeneous population of at least six receptor subtypes, called Y₁–y₆ receptors. All of them are cloned⁵ except the Y₃ receptor. The Y receptors belong to the rhodopsin-like super-family of the G protein coupled receptors. Upon activation they inhibit the adenylate cyclase and lead to decreased levels of intracellular calcium. Whereas NPY is a ligand displaying nanomolar affinities at all Y receptors, PP is relatively selective for the Y₄ receptor with published K_D values ranging from 0.03 to 0.26 nM.6., 7. It also possesses (although slightly reduced) affinity to the Y₅ receptor (IC₅₀ value of 58 nM measured for hPP in a competition assay with hNPY) but virtually no affinity to the Y₁ and Y₂ receptor.⁸ NPY exhibits more than 100-fold lower affinity towards the Y₄ receptor.⁹ Hence, PP presents an interesting peptide to yield information about structural requirements responsible for subtype-specificity at the Y₄ receptor. See Table 1 for the amino acid sequences of the peptides discussed here.

The crystal structure of avian PP (aPP) was published by Blundell et al. in 1981 and presented one of the first high-resolution structures for smaller peptides.¹⁰ It is also the only one available for members of the NPY family. aPP possesses an elongated shape and contains two regions of secondary structure: at the N terminus an extended type II polyproline helix comprising residues 1–8, which is connected by a β-turn to the C-terminal α-helix extending from residue 14 to 31. The polyproline helix is bent back onto the C-terminal α-helix and the interface in between consists of hydrophobic contacts. This structural motif has been named the PP-fold and was later confirmed by the solution structure of bPP elucidated by NMR.¹¹ In the latter structure the N-terminal residues 4–8 clearly associate with the C-terminal α-helix (residues 15–32). The four residues at both the C terminus and the N terminus are poorly defined in solution. The fact that such a small peptide is capable of forming a stable secondary structure in solution is remarkable and therefore attempts have been made to use the aPP skeleton as a basis to create peptides with defined folds.12., 13., 14. Interestingly, the crystal structure presented a dimer linked via Zn²⁺, in which the two monomer subunits associated such that the resulting dimer has 2-fold symmetry. In solution, additional nuclear Overhauser effects (NOEs) were found that might have been due to intermolecular interactions, however, the data did not allow them to decide unambiguously on the aggregation state.

Since NPY and PP share rather high sequence-homology (approx. 50%), and because both molecules possess an amphiphilic helix, such a PP-fold was originally also postulated for hNPY.¹⁵ However, later structures for hNPY¹⁶ and pNPY¹⁷ revealed NPY dimers in solution, in which the N termini remain free and flexible. The dimerization interface was postulated to comprise residues from the α-helices. We could demonstrate by using a mixture of ¹⁵N uniformly labeled pNPY and a mutant of pNPY, in which Gln34 was replaced by the spin-label containing amino acid TOAC, that pNPY forms a dynamic equilibrium of dimers in solution in which the monomers associate via parallel or anti-parallel alignment of the helices.¹⁸

There is accumulating evidence that many hormones display amphiphilic secondary structures, mostly α-helices. In their pioneering work Kaiser & Kézdy¹⁹ could demonstrate the importance of an amphiphilic helix for biological activity by investigating a series of mutant peptides. They suggested that an amphiphilic environment represented by the membrane–water interface could impose secondary structure to a peptide, which is otherwise unstructured in aqueous solution. Moreover, they postulated that interactions of these peptides with their receptor macromolecules are formed by only a few key residues. Later on, their view was refined by Schwyzer in his membrane compartment model20., 21. and by Moroder.²² Again, their theory postulates that the membrane induces a conformation that is partially preformed for receptor recognition. Importantly, it is the membrane-bound conformation that is recognized initially by the receptor. For the case of the polypeptide PACAP Inooka et al.²³ could recently demonstrate that only little conformational difference between receptor-bound conformation as inferred from transfer NOE data and the micelle-bound state exists. Accordingly, we have determined the structures of pNPY,¹⁸ [Ala31,Pro32]-NPY²⁴ and bPP when bound to DPC micelles. In our previous work we could show that side-chains of the hydrophobic residues of the amphiphilic helix were intercalating into the hydrophobic interior of the micelles, thereby anchoring pNPY such that the C-terminal helix is parallel with the membrane surface. From this work, the question arose of whether the hydrophobic contacts between the α-helix and the polyproline helix in the pancreatic polypeptide would be relinquished and new contacts with the micelle be formed instead. Moreover, bPP and pNPY target similar receptors, though with very different affinity to the different receptor subtypes, and at least for pNPY it is known that residues of the C-terminal pentapeptide are important for forming contacts to the receptor. Hence, it is of great interest whether any structural differences in the conformations of the membrane-bound species between pNPY and bPP exist in that part of the molecules.

Section snippets

Resonance assignments

Resonance assignments of the ¹⁵N–¹H correlation map of unligated bPP were mainly performed from an ¹H NOE-relayed [¹⁵N,¹H]-HSQC spectrum. Especially for amide moieties from the C-terminal α-helical segment, sequential NOEs between amide protons largely facilitated the analysis. Assignments in the N-terminal part relied upon observation of sequential H^NH^α NOEs and the proton chemical shifts as published by Li et al.¹¹ The proton chemical shifts agreed very well with the published data.

Resonance

Discussion

In our investigations we focussed on two aspects of the molecular structure of bPP. Firstly, we were interested to know whether the PP fold would also exist in the membrane-bound state. Secondly, any changes of the conformation in the C-terminal pentapeptide would be remarkable in the discussion of structure–activity relationships at the Y receptors. The back-folding of the N-terminal polyproline helix onto the C-terminal α-helix was established by Li et al.¹¹ from the observation of long-range

Cloning, expression and purification of ¹⁵N-labeled bovine PP (bPP)

For the production of ¹⁵N-labeled bPP, we followed the strategy described by Khono et al.⁶⁹ Following his approach, the peptide was expressed as the N-terminally decahistidine-tagged yeast ubiquitin fusion-protein, from which bPP was liberated enzymatically with ubiquitin hydrolase (YUH). C-terminal amidation was performed by enzymatic conversion of an extra Gly residue into an amide function using the α-amidating peptidyl glycine amidase (PAM). To generate a DNA-template, overlapping primers

Acknowledgements

Financial support from the ETH Zürich (grants 0 20 439-97 (to R.B.) and TH-39/00-3 (to M.L.) is kindly acknowledged. We thank Professor A.G. Beck-Sickinger for general interest and helpful discussions. We further acknowledge software support by Dr P. Güntert and thank Dr T. Khono, who supplied us with the plasmids pUBK19 and pYUHK20b.

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Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) form the evolutionarily conserved pancreatic polypeptide family. While the fold is widely utilized in nature, crystal structures remain elusive, particularly for the human forms, with only the structure of a distant avian form of PP reported. Here we utilize a crystallization chaperone (antibody Fab fragment), specifically recognizing the amidated peptide termini, to solve the structures of human NPY and human PYY. Intriguingly, and despite limited sequence identity (~50%), the structure of human PYY closely resembles that of avian PP, highlighting the broad structural conservation of the fold throughout evolution. Specifically, the PYY structure is characterized by a C-terminal amidated α-helix, preceded by a backfolded poly-proline N-terminus, with the termini in close proximity to each other. In contrast, in the structure of human NPY the N-terminal component is disordered, while the helical component of the peptide is observed in a four-helix bundle type arrangement, consistent with a propensity for multimerization suggested by NMR studies.
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Non-specific binding of Y receptor agonists to intact CHO cells, and to CHO cell or rat brain particulates, is much greater for human neuropeptide Y (hNPY) compared to porcine peptide Y (pPYY), and especially relative to human pancreatic polypeptide (hPP). This binding of hNPY is reduced by alkali cations in preference to non-ionic chaotrope urea, while the much lower non-specific binding of pPYY is more sensitive to urea. The difference could mainly be due to the 10–16 stretch in 36-residue Y agonists (residues 8–14 in N-terminally clipped 34-peptides), located in the sector that contains all acidic residues of physiological Y agonists. Anionic pairs containing aspartate in the 10–16 zone could be principally responsible for non-specific attachments, but may also aid the receptor site binding. Two such pairs are found in hNPY, one in pPYY, and none in hPP. The hydroxyl amino acid residue at position 13 in mammalian PYY and PP molecules could lower conformational plasticity and the non-selective binding via intrachain hydrogen bonding. The acidity of this tract could also be important in agonist selectivity of the Y receptor subtypes. The differences point to an evolutionary reduction of promiscuous protein binding from NPY to PP, and should also be important for Y agonist selectivity within NPY receptor group, and correlate with partial agonism and out-of group cross-reactivity with other receptors.
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^†: Present address: R. Bader, Department of Physics, Biological Physics, Cavendish Laboratory, University of Cambridge, Madingley Road, Cambridge CB3 0HE, UK.

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Journal of Molecular Biology

Bovine Pancreatic Polypeptide (bPP) Undergoes Significant Changes in Conformation and Dynamics upon Binding to DPC Micelles

Abstract

Introduction

Section snippets

Resonance assignments

Discussion

Cloning, expression and purification of 15N-labeled bovine PP (bPP)

Acknowledgements

Isolation and biogenesis of a new peptide from pancreatic islets

Nature

Roles of pancreatic polypeptide in regulation of food intake

Peptides

Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats

Endocrinology

Neuropeptide regulation of feeding in dogs

Am. J. Physiol.

XVI International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors

Pharmacol. Rev.

Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands

Peptides

Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted desensitization

J. Pharmacol. Exp. Ther.

Y-receptor affinity modulation by the design of pancreatic polypeptide/neuropeptide Y chimera led to Y(5)-receptor ligands with picomolar affinity

Peptides

Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family

J. Pept. Sci.

X-ray analysis (1.4 Å resolution) of avian pancreatic polypeptide: small globular protein hormone

Proc. Natl Acad. Sci. USA

Sequence-specific 1H NMR assignments and solution structure of bovine pancreatic polypeptide

Biochemistry

Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display

Bioorg. Med. Chem. Letters

Highly specific DNA recognition by a designed miniature protein

J. Am. Chem. Soc.

Concerted evolution of structure and function in a miniature protein

J. Am. Chem. Soc.

Solution conformation of human neuropeptide Y by 1H nuclear magnetic resonance and restrained molecular dynamics

Eur. J. Biochem.

Solution structure of human neuropeptide Y

J. Biomol. NMR

Structure of neuropeptide Y dimer in solution

Eur. J. Biochem.

Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection

J. Mol. Biol.

Amphiphilic secondary structure: design of peptide hormones

Science

Membrane lipid phase as catalyst for peptide–receptor interactions

Proc. Natl Acad. Sci. USA

Molecular mechanism of opioid receptor selection

Biochemistry

New evidence for a membrane-bound pathway in hormone receptor binding

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Nature Struct. Biol.

Key motif to gain selectivity at the neuropeptide Y5-receptor: solution structure and dynamics of [Ala31,Pro32]-NPY

Biochemistry

NMR of Proteins and Nucleic Acids

Torsion angle dynamics for NMR structure calculation with the new program Dyana

J. Mol. Biol.

AMBER 6

AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energectic properties of molecules

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Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Computation of sterically allowed proton–proton distances and statistical analysis of proton–proton distances in single crystal protein conformations

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Analysis of the backbone dynamics of interleukin-1 beta using two- dimensional inverse detected heteronuclear 15N–1H NMR spectroscopy

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Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease

Cloning, expression and purification of ¹⁵N-labeled bovine PP (bPP)

Sequence-specific ¹H NMR assignments and solution structure of bovine pancreatic polypeptide

Solution conformation of human neuropeptide Y by ¹H nuclear magnetic resonance and restrained molecular dynamics

Sequential resonance assignments in protein ¹H nuclear magnetic resonance spectra. Computation of sterically allowed proton–proton distances and statistical analysis of proton–proton distances in single crystal protein conformations

Analysis of the backbone dynamics of interleukin-1 beta using two- dimensional inverse detected heteronuclear ¹⁵N–¹H NMR spectroscopy

Backbone dynamics of proteins as studied by ¹⁵N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease

Motional model analysis of protein and peptide dynamics using ¹³C and ¹H NMR relaxation