Complex with a phage display-derived peptide provides insight into the function of insulin-like growth factor I

The dramatic improvement in the NMR spectra of insulin-like growth factor I (IGF-I) in the presence of a peptide identified from a phage display library has allowed for the first time the determination of a high-resolution solution structure for much of IGF-I. The three helices of IGF-I in this complex have an arrangement similar to that seen in high-resolution crystal structures of IGF-I and insulin, although there are differences in the conformation and precise location of helix 3. A cluster of hydrophobic and basic side chains within the turn-helix motif of the peptide contact a hydrophobic patch on helices 1 and 3 of IGF-I. The importance of this patch for tight binding was verified using alanine scanning mutagenesis of the peptide in two different phage display formats. Consistent with its antagonistic activity, the peptide binds to a region implicated by mutagenesis studies to be important for association with IGF binding proteins (IGFBPs). The ability of the peptide to also inhibit signaling has important implications for the manner in which IGF-I interacts with its receptor. Interestingly, the peptide uses the same binding site as detergent and a fragment of IGFBP-5 identified in other IGF-I complexes. The ligand-induced structural variability of helix 3 in these complexes suggests that exchange between such conformations may be the source of the dynamic nature of free IGF-I and likely has functional significance for the ability of IGF-I to recognize two signaling receptors and six binding proteins with high affinity.     

Total alanine-scanning mutagenesis of insulin-like growth factor I (IGF-I) identifies differential binding epitopes for IGFBP-1 and IGFBP-3.

The bioavailability of insulin-like growth factor I (IGF-I) in the serum and tissues is controlled by members of the IGF binding protein family (IGFBP). These proteins form high-affinity complexes with IGF-I and thereby either inhibit or potentiate its mitogenic and metabolic effects. Thus, understanding the IGF-IGFBP interaction at the molecular level is crucial for attempts to modulate IGF-I activity in vivo. We have systematically investigated the binding contribution of each IGF-I amino acid side chain toward IGFBP-1 and IGFBP-3, combining alanine-scanning mutagenesis and monovalent phage display. Surprisingly, most IGF-I residues could be substituted by alanines, resulting in less than 5-fold affinity losses for IGFBP-3. In contrast, binding of IGFBP-1 was more sensitive to alanine substitutions in IGF-I. The glutamate and phenylalanine at positions 3 and 49 were identified as major specificity determinants for IGFBP-1: the corresponding alanine mutations, E3A and F49A, selectively decreased IGFBP-1 binding by 34- and 100-fold, whereas IGFBP-3 affinity was not affected or reduced maximally 4-fold. No side chain specificity determinant was found for IGFBP-3. Instead, our results suggest that the N-terminal backbone region of IGF-I is important for binding to IGFBP-3. The fact that the functional binding epitopes on IGF-I are overlapping but distinct for both binding proteins may be exploited to design binding protein-specific IGF variants.     

Conformational and structural characteristics of peptides binding to HLA-DR molecules.

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.     

NMR structural studies of the myristoylated N-terminus of ADP ribosylation factor 6 (Arf6)

Arf proteins are guanine nucleotide binding proteins that are implicated in endocytotic pathways and vesicle trafficking. The two widely studied isoforms of Arf proteins (Arf1 and Arf6) have different cellular functions and localizations but similar structures. Arf proteins have an N-terminal helix with a covalently bound myristoyl group. Except structural models, there are no three dimensional structures of the myristoylated N-terminal peptide or the intact myristoylated Arf proteins. However, understanding the role of both the myristoyl group and the N-terminal helix based on the details of their molecular structures is of great interest. In the solution structure of myristoylated N-terminal peptide of Arf6 described here, the myristoyl group folds toward the N-terminus to interact with the hydrophobic residues in particular, the phenyl ring. Also, the structure of the dodecylphosphocholine (DPC) micelle-bound of the peptide together with paramagnetic studies showed that the myristoyl group is inserted into the micelle while residues V4-G10 interact with the surface of the micelle. The structural differences between the unbound and micelle-bound myristoylated N-terminal peptide of Arf6 involves the myristoyl group and the side chains of the hydrophobic residues.     

Amino acid determinants of beta-hairpin conformation in erythropoeitin receptor agonist peptides derived from a phage display library

Display of peptide libraries on filamentous phage has led to the identification of peptides of the form X(2-5)CX(2)GPXTWXCX(2-5) (where X is a variable residue) that bind to the extra-cellular portion of the erythropoietin receptor (EPO-R). These peptides adopt beta-hairpin conformations when co-crystallized with EPO-R. Solution NMR studies reveal that the peptide is conformationally heterogeneous in the absence of receptor due to cis-trans isomerization about the Gly-Pro peptide bond. Replacement of the conserved threonine residue with glycine at the turn i+3 position produces a stable beta-hairpin conformation in solution, although this peptide no longer has activity in an EPO-R-dependent cell proliferation assay. A truncated form of the EPO-R-binding peptide (containing the i+3 glycine residue) also forms a highly populated, monomeric beta-hairpin. In contrast, phage-derived peptide antagonists of insulin-like growth factor binding protein 1 (IGFBP-1) have a high level of sequence identity with the truncated EPO-R peptide (eight of 12 residues) yet adopt a turn-alpha-helix conformation in solution. Peptides containing all possible pairwise amino acid substitutions between the EPO-R and IGFBP-1 peptides have been analyzed to assess the degree to which the non-conserved residues stabilize the hairpin or helix conformation. All four residues present in the original sequence are required for maximum population of either the beta-hairpin or alpha-helix conformation, although some substitutions have a more dominant effect. The results demonstrate that, within a given sequence, the observed conformation can be dictated by a small subset of the residues (in this case four out of 12).     

Molecular dynamics simulations predict a tilted orientation for the helical region of dynorphin A(1-17) in dimyristoylphosphatidylcholine bilayers

The structural properties of the endogenous opioid peptide dynorphin A(1-17) (DynA), a potential analgesic, were studied with molecular dynamics simulations in dimyristoylphosphatidylcholine bilayers. Starting with the known NMR structure of the peptide in dodecylphosphocholine micelles, the N-terminal helical segment of DynA (encompassing residues 1-10) was initially inserted in the bilayer in a perpendicular orientation with respect to the membrane plane. Parallel simulations were carried out from two starting structures, systems A and B, that differ by 4 A in the vertical positioning of the peptide helix. The complex consisted of approximately 26,400 atoms (dynorphin + 86 lipids + approximately 5300 waters). After >2 ns of simulation, which included >1 ns of equilibration, the orientation of the helical segment of DynA had undergone a transition from parallel to tilted with respect to the bilayer normal in both the A and B systems. When the helix axis achieved a approximately 50 degrees angle with the bilayer normal, it remained stable for the next 1 ns of simulation. The two simulations with different starting points converged to the same final structure, with the helix inserted in the bilayer throughout the simulations. Analysis shows that the tilted orientation adopted by the N-terminal helix is due to specific interactions of residues in the DynA sequence with phospholipid headgroups, water, and the hydrocarbon chains. Key elements are the "snorkel model"-type interactions of arginine side chains, the stabilization of the N-terminal hydrophobic sequence in the lipid environment, and the specific interactions of the first residue, Tyr. Water penetration within the bilayer is facilitated by the immersed DynA, but it is not uniform around the surface of the helix. Many water molecules surround the arginine side chains, while water penetration near the helical surface formed by hydrophobic residues is negligible. A mechanism of receptor interaction is proposed for DynA, involving the tilted orientation observed from these simulations of the peptide in the lipid bilayer.     

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.     

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.     

Addition of side chain interactions to modified Lifson-Roig helix-coil theory: application to energetics of phenylalanine-methionine interactions.

We introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson-Roig helix-coil theory of Doig et al. (1994, Biochemistry 33:3396-3403). The helix/coil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields delta G for the Phe-Met orientation to be -0.75 kcal.mol-1, whereas that for the Met-Phe orientation is -0.54 kcal.mol-1. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe-Met i, i + 4 at the N-terminus, the C-terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain-side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix.     

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.     

Conformational and biochemical analysis of the cyclic peptides which modulate serine protease activity.

Prostate-specific antigen (PSA), a member of the kallikrein sub-group of the trypsin serine protease family, is a widely used marker for prostate cancer. Several sequences with specific binding to PSA have been identified by using phage display peptide libraries. The GST-fusion proteins of the characterized sequences have been shown to increase the enzyme activity of PSA to a synthetic substrate. The corresponding three cyclic synthetic analogues CVFTSNYAFC (A-1), CVFAHNYNYLVC (B-2) and CVAYCIEHHCWTC (C-4) have similar PSA promoting activity. Despite differences in the amino acid sequences, all three peptides bind to the same region of PSA. The conformation of the peptides was investigated by proton NMR spectroscopy. In addition, alanine replacement was used to characterize the prerequisites for binding. It is proposed that interactions with PSA are based on the aromatic and hydrophobic features of the amino acid side chains. Furthermore, it is suggested that peptides form beta-turn structures forced by cysteine bridges directing important aromatic side chains to the same side of the turn-structure.     

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.     

The interaction of insulin-like growth factor-I with the N-terminal domain of IGFBP-5

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.     

Binding and toxicity of apamin. Characterization of the active site

The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln-17 and the side chains of Arg-13 and Arg-14 (besides the charge effects). The interaction of apamin with its receptor, a calcium-activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.     

Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1)

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.     

A new mechanism for metal ion-assisted interchain helix assembly in a naturally occurring peptide mediated by optimally spaced gamma-carboxyglutamic acid residues

Helix-helix interactions, such as those that occur in coiled-coil domains, four-helix bundles, or membrane-spanning helical bundles, are important to the structural organization and function of numerous proteins. However, tractable peptide models for studying such structural elements have been limited to synthetic analogs of coiled-coil protein domains and de novo designed peptides. The present study provides evidence that conantokin-G (con-G), a gamma-carboxyglutamate (Gla)-rich neuroactive peptide from a venomous marine snail, can self-associate in the presence of certain divalent metal cations. Sedimentation equilibrium analyses of con-G show that Ca2+ binding promotes peptide dimerization, while the addition of the tighter binding divalent cations, Mg2+, Zn2+, and Mn2+, does not result in intermolecular association. The effects of specific residue replacements indicate that an i, i + 4, i + 7, i + 11 arrangement of Gla residues is essential for con-G self-assembly. To determine the relative chain orientation of the dimeric assembly, distributions of Cys-containing con-G variants were examined in thiol-disulfide rearrangement assays and the results were consistent with an antiparallel alignment. Our data suggest that the driving force for con-G dimerization stems from the appropriate balance of interchain and intrachain metal ion coordination by Gla residues in similar locations. These findings suggest a new role for Gla residues and accompanying cation binding in the stabilization of interstrand helix association in a natural product and provide a model for controlled assembly of peptide chains or segments of larger proteins.     

Stabilizing nonpolar/polar side-chain interactions in the alpha-helix.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in alpha-helices, can in fact be stabilizing. Residues spaced i, i + 4 in alpha-helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile-Lys, Ile-Arg, and Val-Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix-coil theory to calculate the free energy for the interactions. All three stabilize the helix with DeltaG between -0.14 and -0.32 kcal x mol(-1). The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues.     

Thrombin receptor-activating peptides (TRAPs): investigation of bioactive conformations via structure-activity, spectroscopic, and computational studies

The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extracellular N-terminus to expose an agonist peptide ligand, which is tethered to the receptor itself. Synthetic peptides containing the agonist motif, such as SFLLRN for human PAR-1, are capable of causing full receptor activation. We have probed the possible bioactive conformations of thrombin receptor-activating peptides (TRAPs) by systematic introduction of certain conformational perturbations, involving alpha-methyl, ester psi(COO), and reduced-amide psi(CH2N) scans, into the minimum-essential agonist sequence (SFLLR) to probe the importance of the backbone conformation and amide NH hydrogen bonding. We performed extensive conformational searches of representative pentapeptides to derive families of putative bioactive structures. In addition, we employed 1H NMR and circular dichroism (CD) to characterize the conformational disposition of certain pentapeptide analogues experimentally. Activation of platelet aggregation by our pentapeptide analogues afforded a structure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of peptide ligands for the thrombin receptor independent of a functional cellular response derived from receptor activation (i.e. a pure molecular recognition event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topography relative to platelet functional activity and PAR-1 binding. The absolute requirement of a positively charged N-terminus for strong agonist activity was contradicted by the N-terminal hydroxyl peptide psi(HO)S-FLLR-NH2. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be involved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that the amide nitrogen at this position can be substituted by an oxygen, as in SF-psi(COO)-LLR-NH2, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH2 was active, whereas SF-NMeL-LR-NH2 was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2-aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A and 5B lack agonist activity. On the basis of our results, we suggest that an extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction).     

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.     

Design and synthesis of 3alpha-helix peptides forming a cavity for a fluorescent ligand

As a model of receptor protein, a series of 3alpha-helix bundle peptides constructed on a template peptide were designed so as to possess a hydrophobic cavity. The size of cavity was modulated by simple replacements of Leu residues to Ala residues in the hydrophobic core. Binding abilities to 8-anilino-1-naphthalenesulfonic acid (ANS) were estimated by the increase of fluorescence intensity. The peptide having three or four Ala residues in the hydrophobic core remarkably increased the binding ability for ANS, though the peptide having two Ala residues gave an inefficient cavity for ANS. The peptide having six Ala residues decreased the binding ability due to crucial destabilization of the helix bundle structure. This scaffold can be utilized to a receptor model, while further tuning of the sequence is necessary.