First observation of solution structures of bradykinin–penta-O-galloyl-d-glucopyranose complexes as determined by NMR and simulated annealing

Polyphenols (tannins) are known for their high propensity to precipitate proteins. They bind most strongly to proteins with a high proline content. Understanding the mechanism of this association is of prime interest because this interaction might induce protein conformational changes that may modify their biological activity. To investigate the interaction, an NMR study was carried out on the binding of a representative polyphenol, penta-O-galloyl-d-glucopyranose, to a nonapeptide hormone, bradykinin (BDK), where proline accounts for 30% of residues. Series of 1D and 2D-NMR experiments were performed. For the first time, a three-dimensional structure of complexes was determined using 2D-NMR experiments and molecular modeling. These structure calculations are a potent tool to understand how the association arises. They clearly show that the interaction is a complex phenomenon where several parameters are involved. The PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. Proline and arginine are good anchoring points and the glycine gives a certain flexibility in the peptide backbone that allows the polyphenol to approach and interact. Therefore, it is not only the hydrophobic stackings between galloyl rings and proline and hydrogen bonding involving arginine and aromatic rings which are important. The residue sequence and the side chain steric bulk also intervene.     

NMR characterization of monomeric and oligomeric conformations of human calcitonin and its interaction with EGCG

Calcitonin is a 32-residue peptide hormone known for its hypocalcemic effect and its inhibition of bone resorption. While calcitonin has been used in therapy for osteoporosis and Paget's disease for decades, human calcitonin (hCT) forms fibrils in aqueous solution that limit its therapeutic application. The molecular mechanism of fiber formation by calcitonin is not well understood. Here, high-resolution structures of hCT at concentrations of 0.3 mM and 1 mM have been investigated using NMR spectroscopy. Comparing the structures of hCT at different concentrations, we discovered that the peptide undergoes a conformational transition from an extended to a β-hairpin structure in the process of molecular association. This conformational transition locates the aromatic side chains of Tyr12 and Phe16 in a favorable way for intermolecular π-π stacking, which is proposed to be a crucial interaction for peptide association and fibrillation. One-dimensional (1)H NMR experiments confirm that oligomerization of hCT accompanies the conformational transition at 1 mM concentration. The effect of the polyphenol epigallocatechin 3-gallate (EGCG) on hCT fibrillation was also investigated by NMR and electron microscopy, which show that EGCG efficiently inhibits fibril formation of hCT by preventing the initial association of hCT before fiber formation. The NMR experiments also indicate that the interaction between aromatic rings of EGCG and the aromatic side chains of the peptide may play an important role in inhibiting fibril formation of hCT.     

Peptide backbone conformation affects the substrate preference of protein arginine methyltransferase I

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.     

Recognition of accessory protein motifs by the gamma-adaptin ear domain of GGA3

Adaptor proteins load transmembrane protein cargo into transport vesicles and serve as nexuses for the formation of large multiprotein complexes on the nascent vesicles. The gamma-adaptin ear (GAE) domains of the AP-1 adaptor protein complex and the GGA adaptor proteins recruit accessory proteins to these multiprotein complexes by binding to a hydrophobic motif. We determined the structure of the GAE domain of human GGA3 in complex with a peptide based on the DFGPLV sequence of the accessory protein Rabaptin-5 and refined it at a resolution of 2.2 A. The leucine and valine residues of the peptide are partly buried in two contiguous shallow, hydrophobic depressions. The anchoring phenylalanine is buried in a deep pocket formed by the aliphatic portions of two conserved arginine residues, along with an alanine and a proline, illustrating the unusual function of a cluster of basic residues in binding a hydrophobic motif.     

Recognition characters in peptide–polyphenol complex formation

Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein–polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.     

Physical association of GPR54 C-terminal with protein phosphatase 2A

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.     

The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC.

Cell surface complexes of class I MHC molecules and bound peptide antigens serve as specific recognition elements controlling the cytotoxic immune response. The 2.1 A structure of the human class I MHC molecule HLA-B27 provides a detailed composite image of a co-crystallized collection of HLA-B27-bound peptides, indicating that they share a common main-chain structure and length. It also permits direct visualization of the conservation of arginine as an "anchor" side chain at the second peptide position, which is bound in a potentially HLA-B27-specific pocket and may therefore have a role in the association of HLA-B27 with several diseases. Tight peptide binding to class I MHC molecules appears to result from the extensive contacts found at the ends of the cleft between peptide main-chain atoms and conserved MHC side chains, which also involve the peptide in stabilizing the three-dimensional fold of HLA-B27. The concentration of binding interactions at the peptide termini permits extensive sequence (and probably some length) variability in the center of the peptide, where it is exposed for T cell recognition.     

The molecular interaction of human salivary histatins with polyphenolic compounds.

Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst-polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1-mM dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.     

Recognition characters in peptide-polyphenol complex formation

Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein-polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.     

Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

Molecular basis for autoregulatory interaction between GAE domain and hinge region of GGA1

Golgi-localizing, gamma-adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The gamma-adaptin ear (GAE) domain of GGAs and the gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.     

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ(XP), contrary to conventional thinking that positive Γ(XP)'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ(XP), which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation.     

Rings and ribbons in protein structures: Characterization using helical parameters and Ramachandran plots for repeating dipeptides

Helical parameters displayed on a Ramachandran plot allow peptide structures with successive residues having identical main chain conformations to be studied. We investigate repeating dipeptide main chain conformations and present Ramachandran plots encompassing the range of possible structures. Repeating dipeptides fall into the categories: rings, ribbons, and helices. Partial rings occur in the form of “nests” and “catgrips”; many nests are bridged by an oxygen atom hydrogen bonding to the main chain NH groups of alternate residues, an interaction optimized by the ring structure of the nest. A novel recurring feature is identified that we name unpleated β, often situated at the ends of a β‐sheet strand. Some are partial rings causing the polypeptide to curve gently away from the sheet; some are straight. They lack β‐pleat and almost all incorporate a glycine. An example is the first glycine in the GxxxxGK motif of P‐loop proteins. Ribbons in repeating dipeptides can be either flat, as seen in repeated type II and type II′ β‐turns, or twisted, as in multiple type I and type I′ β‐turns. Hexa‐ and octa‐peptides in such twisted ribbons occur frequently in proteins, predominantly with type I β‐turns, and are the same as the “β‐bend ribbons” hitherto identified only in short peptides. One is seen in the GTPase‐activating protein for Rho in the active, but not the inactive, form of the enzyme. It forms a β‐bend ribbon, which incorporates the catalytic arginine, allowing its side chain guanidino group to approach the active site and enhance enzyme activity. Proteins 2014; 82:230–239. © 2013 Wiley Periodicals, Inc.     

Structural features of apolipoprotein B synthetic peptides that inhibit lipoprotein(a) assembly

Lipoprotein(a) [Lp(a)] is assembled via an initial noncovalent interaction between apolipoprotein B100 (apoB) and apolipoprotein(a) [apo(a)] that facilitates the formation of a disulfide bond between the two proteins. We previously reported that a lysine-rich, alpha-helical peptide spanning human apoB amino acids 4372-4392 was an effective inhibitor of Lp(a) assembly in vitro. To identify the important structural features required for inhibitory action, new variants of the apoB4372-4392 peptide were investigated. Introduction of a central leucine to proline substitution abolished the alpha-helical structure of the peptide and disrupted apo(a) binding and inhibition of Lp(a) formation. Substitution of hydrophobic residues in the apoB4372-4392 peptide disrupted apo(a) binding and inhibition of Lp(a) assembly without disrupting the alpha-helical structure. Substitution of all four lysine residues in the peptide with arginine decreased the IC50 from 40 microM to 5 microM . Complexing of the arginine-substituted peptide to dimyristoylphosphatidylcholine improved its activity further, yielding an IC50 of 1 microM. We conclude that the alpha-helical structure of apoB4372-4392, in combination with hydrophobic residues at the lipid/water interface, is crucial for its interaction with apo(a). Furthermore, the interaction of apoB4372-4392 with apo(a) is not lysine specific, because substitutions with arginine result in a more effective inhibitor.     

Solvent interactions with the Trp-cage peptide in 35% ethanol-water

Intermolecular NOE experiments have been used to explore interactions of water and ethanol molecules in 35% ethanol/65% water (v/v) with the peptide Trp-cage at temperatures from 5 to 25 degrees C. Magnetic dipole-dipole cross-relaxation terms sigma(HH) (NOE) and sigma(HH) (ROE) for interaction of solvent components with spins of the peptide suggest that ethanol molecules associate with backbone atoms for times of the order of nanoseconds at 5 degrees C. Formation of peptide-ethanol complexes can also account for the larger-than-expected values of cross-relaxation terms at higher temperatures. Hydrocarbon side chains of the peptide do not appear to experience such interactions with ethanol. Cross relaxation resulting from water-peptide interactions are consistent with long-lived water interactions with the backbone atoms. Water cross relaxation with nonpolar side chains of the peptide (Leu2, Ile4, Leu7, and proline residues) are only those expected for bulk solvent. However, long-lived association of both water and ethanol with the polar side chains of Tyr3 and Trp6 is indicated by the data. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 862-872, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Spectra and structure of complexes formed by sodium fusidate and potassium helvolate with beta- and gamma-cyclodextrin

The complexation of two steroid antibiotics of the fusidane family, sodium fusidate and potassium helvolate, by beta-CD and gamma-CD has been studied by using 1D and 2D-NMR techniques. Both guests form 1:1 complexes with gamma-CD and 1:2 (guest:cyclodextrin) complexes with beta-CD. Thus, both antibiotics behave as monotopic and ditopic guests when they are complexed by gamma-CD and beta-CD, respectively. Both steroids enter into the cavity of the gamma-CD by the side chain, reaching the central region of the steroid (rings C and D), whereas the A and B (partially) rings remain outside. For beta-CD complexes, ROESY spectra show a remarkable absence of interactions of the protons of the C and D rings, whereas clear interactions corresponding to the side chain, and A and B rings are observed. The obtained equilibrium constants (see previous paper) are discussed in terms of the structures proposed for the complexes. NMR spectra of sodium fusidate are revised, and a full assignment of the 1H and 13C NMR spectra is presented for potassium helvolate.     

Trypsin cleaves exclusively C-terminal to arginine and lysine residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.     

Effect of different arginine methylations on the thermodynamics of Tat peptide binding to HIV-1 TAR RNA

RNA-binding proteins are an important class of mediators that regulate cell function and differentiation. Methylation of arginine, a post-translational modification (PTM) found in these proteins, can modulate their function. Arginine can be monomethylated or dimethylated, depending on the type of methyl transferases involved. This paper describes a comparative study of the thermodynamics of unmodified and modified Tat peptide interaction with TAR RNA, where the peptide is methylated at epsilon (?) and eta (η) nitrogen atoms of guanidinium group of arginine side chain at position 52 or 53. The results indicate that monomethylation of arginine at epsilon (?) nitrogen atom enhances binding affinity, owing to a more favourable enthalpy component which overrides the less favourable entropy change. In contrast, monomethylation of arginine residue at η nitrogen results in reduced binding affinity originating exclusively from a less favourable enthalpy change leaving entropic component unaffected. However, in case of simultaneous methylation at ? and η positions, the binding parameters remain almost unaffected, when compared to the unmodified peptide. In case of symmetric dimethylation at η position the observed enthalpy change of the binding was found to be smaller than the values obtained for the unmodified peptide. Asymmetric dimethylation at η position showed the most reduced binding affinities owing to less favourable enthalpy changes. These results provide insights that enable elucidation of the biological outcome of arginine methylation as PTMs that regulate protein function, and will contribute to our understanding of how these PTMs are established in vitro and in vivo.     

The role of glycine and prolines in connective tissue fiber staining with hydrogen bonding dyes

Extensive hydrogen bonding of dyes to connective tissue fibers is made possible by the high content of the amino acids proline and glycine in elastin and collagens. Proline confers an extended polypeptide structure and glycine is the only amino acid whose specific side group, -H, is so small that it forms no obstacle to hydrogen bonding between the peptide group and external molecules. Thus, a high proportion of the peptide groups in fibrous proteins are directly accessible to hydrogen bonding groups dye molecules.