Effects of chain length and N-methylation on a cation-pi interaction in a beta-hairpin peptide

The effects of N-methylation and chain length on a cation-pi interaction have been investigated within the context of a beta-hairpin peptide. Significant enhancement of the interaction and structural stabilization of the hairpin have been observed upon Lys methylation. Thermodynamic analysis indicates an increased entropic driving force for folding upon methylation of Lys residues. Comparison of lysine to analogues ornithine (Orn) and diaminobutyric acid (Dab) indicates that lysine provides the strongest cation-pi interaction and also provides the most stable beta-hairpin due to a combination of side chain-side chain interactions and beta-sheet propensities. These studies have significance for the recognition of methylated lysine in histone proteins.     

Structural aspects for the recognition of ATP by ribonucleopeptide receptors

A modular structure of ribonucleopeptide (RNP) affords a framework to construct macromolecular receptors and fluorescent sensors. We have isolated ATP-binding RNP with the minimum of nucleotides for ATP binding, in which the RNA consensus sequence is different from those reported for RNA aptamers against the ATP analogues. The three-dimensional structure of the substrate-binding complex of RNP was studied to understand the ATP-binding mechanism of RNP. A combination of NMR measurements, enzymatic and chemical mapping, and nucleotide mutation studies of the RNP-adenosine complex show that RNP interacts with the adenine ring of adenosine by forming a U:A:U triple with two invariant U nucleotides. The observed recognition mode for the adenine ring is different from those of RNA aptamers for ATP derivatives reported previously. The RNP-adenosine complex is folded into a particular structure by formation of the U:A:U triple and a Hoogsteen type A:U base pair. This recognition mechanism was successfully utilized to convert the substrate-binding specificity of RNP from ATP- to GTP-binding with a C(+):G:C triple recognition mode.     

Formation of an amino-acid-binding pocket through adaptive zippering-up of a large DNA hairpin loop

Background: In vitro selection has identified DNA aptamers that target cofactors, amino acids, peptides and proteins. Structure determination of such ligand-DNA aptamer complexes should elucidate the details of adaptive DNA structural transitions, binding-pocket architectures and ligand recognition. We have determined the solution structure of the complex of a DNA aptamer containing a guanine-rich 18-residue hairpin loop that binds l-argininamide with ∼ 100μM affinity.Results: The DNA aptamer generates its l-argininamide-binding pocket by adaptive zippering up the 18-residue loop through formation of Watson-Crick pairs, mismatch pairs and base triples, while maximizing stacking interactions. Three of the four base triples involve minor-groove recognition through sheared G·A mismatch formation. The unique fold is also achieved through positioning of an adenine residue deep within the minor groove and through nestling of a smaller loop within the larger loop on complex formation. The accessibility to the unique l-argininamide-binding pocket is restricted by a base pair that bridges across one side of the major-groove-binding site. The guanidinium group of the bound l-argininamide aligns through intermolecular hydrogen-bond formation with the base edges of nonadjacent guanine and cytosine residues while being sandwiched between the planes of nonadjacent guanine residues.Conclusions: The available structures of l-arginine/l-argininamide bound to their DNA and RNA targets define the common principles and patterns associated with molecular recognition, as well as the diversity of intermolecular hydrogen-bonding alignments associated with the distinct binding pockets.     

The role of cation-pi interactions in biomolecular association. Design of peptides favoring interactions between cationic and aromatic amino acid side chains.

Cation-pi interactions between amino acid side chains are increasingly being recognized as important structural and functional features of proteins and other biomolecules. Although these interactions have been found in static protein structures, they have not yet been detected in dynamic biomolecular systems. We determined, by (1)H NMR spectroscopic titrations, the energies of cation-pi interactions of the amino acid derivative AcLysOMe (1) with AcPheOEt (2) and with AcTyrOEt (3) in aqueous and three organic solvents. The interaction energy is substantial; it ranges from -2.1 to -3.4 kcal/mol and depends only slightly on the dielectric constant of the solvent. To assess the effects of auxiliary interactions and structural preorganization on formation of cation-pi interactions, we studied these interactions in the association of pentapeptides. Upon binding of the positively-charged peptide AcLysLysLysLysLysNH(2) (5) to the negatively-charged partner AcAspAspXAspAspNH(2) (6), in which X is Leu (6a), Tyr (6b), and Phe (6c), multiple interactions occur. Association of the two pentapeptides is dynamic. Free peptides and their complex are in fast exchange on the NMR time-scale, and 2D (1)H ROESY spectra of the complex of the two pentapeptides do not show intermolecular ROESY peaks. Perturbations of the chemical shifts indicated that the aromatic groups in peptides 6b and 6c were affected by the association with 5. The association constants K(A) for 5 with 6a and with 6b are nearly equal, (4.0 +/- 0.7) x 10(3) and (5.0 +/- 1.0) x 10(3) M(-)(1), respectively, while K(A) for 5 with 6c is larger, (8.3 +/- 1.3) x 10(3) M(-)(1). Molecular-dynamics (MD) simulations of the pentapeptide pairs confirmed that their association is dynamic and showed that cation-pi contacts between the two peptides are stereochemically possible. A transient complex between 5 and 6 with a prominent cation-pi interaction, obtained from MD simulations, was used as a template to design cyclic peptides C(X) featuring persistent cation-pi interactions. The cyclic peptide C(X) had a sequence in which X is Tyr, Phe, and Leu. The first two peptides do, but the third does not, contain the aromatic residue capable of interacting with a cationic Lys residue. This covalent construct offered conformational stability over the noncovalent complexes and allowed thorough studies by 2D NMR spectroscopy. Multiple conformations of the cyclic peptides C(Tyr) and C(Phe) are in slow exchange on the NMR time-scale. In one of these conformations, cation-pi interaction between Lys3 and Tyr9/Phe9 is clearly evident. Multiple NOEs between the side chains of residues 3 and 9 are observed; chemical-shift changes are consistent with the placement of the side chain of Lys3 over the aromatic ring. In contrast, the cyclic peptide C(Leu) showed no evidence for close approach of the side chains of Lys3 and Leu9. The cation-pi interaction persists in both DMSO and aqueous solvents. When the disulfide bond in the cyclic peptide C(Phe) was removed, the cation-pi interaction in the acyclic peptide AC(Phe) remained. To test the reliability of the pK(a) criterion for the existence of cation-pi interactions, we determined residue-specific pK(a) values of all four Lys side chains in all three cyclic peptides C(X). While NOE cross-peaks and perturbations of the chemical shifts clearly show the existence of the cation-pi interaction, pK(a) values of Lys3 in C(Tyr) and in C(Phe) differ only marginally from those values of other lysines in these dynamic peptides. Our experimental results with dynamic peptide systems highlight the role of cation-pi interactions in both intermolecular recognition at the protein-protein interface and intramolecular processes such as protein folding.     

Simple cation-pi interaction between a phenyl ring and a protonated amine stabilizes an alpha-helix in water

Cation-pi interactions have been proposed to be important contributors to protein structure and function. In particular, these interactions have been suggested to provide significant stability at the solvent-exposed surface of a protein. We have investigated the magnitude of cation-pi interactions between phenylalanine (Phe) and lysine (Lys), ornithine (Orn), and diaminobutanoic acid (Dab) in the context of an alpha-helix and have found that only the Phe...Orn interaction provides significant stability to the helix, stabilizing it by -0.4 kcal/mol. This interaction energy is in the same range as a salt bridge in an alpha-helix, and equivalent to the recently reported Trp...Arg interaction in an alpha-helix, despite the fact that Trp...guanidinium interactions have been proposed to be stronger than Phe...ammonium interactions. These results indicate that even the simplest cation-pi interaction can provide significant stability to protein structure and demonstrate the subtle factors that can influence the observed interaction energies in designed systems.     

Stair motifs at protein-DNA interfaces: nonadditivity of H-bond, stacking, and cation-pi interactions

At the interface between protein and double-stranded DNA, stair motifs simultaneously involve three different types of pairwise interactions: aromatic base stacking, hydrogen bonding, and cation-pi. The relative importance of these interactions is studied in the stair motif occurring in the 1TC3 crystal structure, which involves an arginine and two stacked guanines, by means of Hartree-Fock (HF) and M?ller-Plesset energy and free energy calculations, including vibrational, rotational, translational contributions, both in a vacuum and various solvents. The results obtained show an anti-cooperative tendency of the HF energy and vibrational free energy terms, and the cooperativity of the rotational, translational, and solvation free energies. Hence, the cooperativity of the stair motif interactions, in the context of protein-DNA recognition, can be viewed as arising from the environment.     

Influence of N-methylation on a cation-pi interaction produces a remarkably stable beta-hairpin peptide

The methylation of lysine in histone tails is a common posttranslational modification that functions in histone-regulated chromatin condensation, with binding of methylated lysine occurring in aromatic pockets on chromodomain proteins. We have synthesized a highly stable 12-residue beta-hairpin peptide that exploits the histone-related cation-pi interaction between a methylated lysine residue and a tryptophan residue. Thermodynamic analysis reveals significant entropic stabilization of the peptide due to methylation of the lysine residue. Chemical denaturation of the peptide demonstrates two-state behavior. In comparison to other reported, highly stable designed beta-hairpins, this peptide is the most thermally stable beta-hairpin reported to date. This study provides insight into the role of Lys methylation in histone proteins and more generally in mediating protein-protein interactions.     

A designed beta-hairpin peptide for molecular recognition of ATP in water

A designed 12-residue beta-hairpin peptide with a diagonal tryptophan (Trp) pair was shown to bind ATP in water through a combination of aromatic and electrostatic interactions. The affinity for ATP was 5800 M-1 (DeltaG approximately -5.0 kcal/mol), a remarkable affinity for a short, structured peptide in water, consisting of entirely natural amino acid residues. Proton NMR measurements indicate that the adenine ring of the nucleotide is intercalated between the diagonal tryptophans in the bound state. Delineation of the contributions to ATP binding to the hairpin suggest that aromatic interactions contribute approximately -1.8 kcal/mol, while individual electrostatic interactions involving the ATP phosphates and positively charged side chains of the hairpin contribute approximately -1 kcal/mol each. The designed beta-hairpin receptor presents a novel minimalist system to investigate the energetic contributions to protein-nucleic acid recognition through the surface of a beta-sheet.     

Cation-pi interactions: an energy decomposition analysis and its implication in delta-opioid receptor-ligand binding

The nature and strength of the cation-pi interaction in protein-ligand binding are modeled by considering a series of nonbonded complexes involving N-substituted piperidines and substituted monocylic aromatics that mimic the delta-opioid receptor-ligand binding. High-level ab initio quantum mechanical calculations confirm the importance of such cation-pi interactions, whose intermolecular interaction energy ranges from -6 to -12 kcal/mol. A better understanding of the electrostatics, polarization, and other intermolecular interactions is obtained by appropriately decomposing the total interaction energy into their individual components. The energy decomposition analysis is also useful for parametrizing existing molecular mechanics force fields that could then account for energetic contributions arising out of cation-pi interactions in biomolecules. The present results further provide a framework for interpreting experimental results from point mutation reported for the delta-opioid receptor.     

Arginine methylation in a beta-hairpin peptide: implications for Arg-pi interactions, DeltaCp(o), and the cold denatured state

Arginine methylation is a common post-translational modification that plays a role in many cellular processes through mediation of protein-protein interactions. There is still a dearth of structural information as to its role in mediating such interactions, but the available data suggest a possible role of cation-pi interactions in the recognition of methylated arginine. Hence, the effect of arginine methylation on its interaction with tryptophan has been investigated within the context of a beta-hairpin peptide. Arginine methylation was found to enhance the stacking interaction between the cationic guanidinium functionality of arginine and the indole ring of tryptophan, resulting in structural stabilization of the hairpin. Thermodynamic analysis reveals more favorable entropy of hairpin folding with arginine methylation, a more negative change in heat capacity for folding, and a modest decrease in enthalpic driving force. This is consistent with enhanced stacking and hydrophobic interactions through increased surface area of the guanidinium moiety and greater delocalization of positive charge. In addition, these peptides exhibit significant cold denaturation, which can be accounted for by the inclusion of an expression of temperature-dependent DeltaC(p) in the thermodynamic analysis.     

Residue-specific pKa determination of lysine and arginine side chains by indirect 15N and 13C NMR spectroscopy: application to apo calmodulin

Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants. We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pKa determination of individual amino acid side chains. The experiments follow the side-chain 15Nzeta (lysine) and 15Nepsilon or 13Czeta (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede 1H-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the 15Nzeta and 15Nepsilon nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D9k, and FKBP12. For apo calmodulin, residue-specific pKa values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification.     

Minimalist protein design: a beta-hairpin peptide that binds ssDNA

A 28-residue beta-hairpin dimer (WKWK)2 with two Trp and two Lys residues on one face of each beta-sheet was shown to form a complex with single-stranded oligonucleotides at low micromolar concentrations. Each beta-hairpin of the dimer contains a cross-strand Trp-Trp pair in a diagonal orientation which has previously been shown to create a cleft for the intercalation of aromatic guests such as adenine (J. Am. Chem. Soc. 2003, 125, 9580). The beta-hairpin dimer binds 5-residue ssDNA sequences 5'-AAAAA-3', 5'-TTTTT-3', and 5'-CCCCC-3' in water with dissociation constants in the range of 12-30 muM. A weak energetic preference for binding to sequence 5'-AAAAA-3' was observed, which is believed to result from stronger stacking interactions between Trp and the adenine base. The interaction of 5'-AAAAA-3' with the Lys and Trp residues of the peptide was evident by NMR, and a 1:1 association was demonstrated. The recognition of an 11-residue ssDNA sequence occurred with a dissociation constant of 3 muM under near-physiological ionic strength and pH, demonstrating that the beta-hairpin dimer binds ssDNA as strongly as many naturally occurring proteins. The salt dependence of the interaction of the 11-residue oligonucleotide with the peptide dimer indicates that Trp-nucleobase stacking interactions contribute about -4 kcal/mol to recognition, which is much greater than the contribution of nonionic interactions in unstructured peptides containing Trp. Moreover, recognition of the ssDNA demonstrated reduced salt dependence relative to the corresponding duplex, resulting in selectivity for ssDNA under high salt conditions. Peptide (WKWK)2 is a relevant mimic of OB-fold (oligonucleotide/oligosaccharide-binding) proteins which bind ssDNA on the surface of a beta-sheet.     

A molecular tweezer for lysine and arginine

Lysine and arginine play a key role in numerous biological recognition processes controlling, inter alia, gene regulation, glycoprotein targeting and vesicle transport. They are also found in signaling peptide sequences responsible, e.g. for bacterial cell wall biosynthesis, Alzheimer peptide aggregation and skin regeneration. Almost none of all artificial receptor structures reported to date are selective and efficient for lysine residues in peptides or proteins. An artificial molecular tweezer is introduced which displays an exceptionally high affinity for lysine (K(a) approximately 5000 in neutral phosphate buffer). It features an electron-rich torus-shaped cavity adorned with two peripheral anionic phosphonate groups. Exquisite selectivity for arginine and lysine is achieved by threading the whole amino acid side chain through the cavity and subsequent locking by formation of a phosphonate-ammonium/guanidinium salt bridge. This pseudorotaxane-like geometry is also formed in small basic signaling peptides, which can be bound with unprecedented affinity in buffered aqueous solution. NMR titrations, NOESY and VT experiments as well as ITC measurements and Monte Carlo simulations unanimously point to an enthalpy-driven process utilizing a combination of van der Waals interactions and substantial electrostatic contributions for a conformational lock. Since DMSO and acetonitrile compete with the amino acid guest inside the cavity, a simple change in the cosolvent composition renders the whole complexation process reversible.     

Cation-pi interactions stabilize the structure of the antimicrobial peptide indolicidin near membranes: molecular dynamics simulations

We implemented molecular dynamics simulations of the 13-residue antimicrobial peptide indolicidin (ILPWKWPWWPWRR-NH2) in dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. In DPC, a persistent cation-pi interaction between TRP11 and ARG13 defined the structure of the peptide near the interface. A transient cation-pi interaction was also observed between TRP4 and the choline group on DPC lipids. We also implemented simulation of a mutant of indolicidin in the DPC micelle where TRP11 was replaced by ALA11. As a result of the mutation, the boat-shaped conformation is lost and the structure becomes significantly less defined. On the basis of this evidence, we argue that cation-pi interactions determine the experimentally measured, well-defined boat-shaped structure of indolicidin. In SDS, the lack of such interactions and the electrostatic binding of the terminal arginine residues to the sulfate groups leads to an extended peptide structure. To the best of our knowledge, this is the first time that a cation-pi interaction between peptide side chains has been shown to stabilize the structure of a small antimicrobial peptide. The simulations are in excellent agreement with available experimental measurements: the backbone of the peptide is more ordered in DPC than in SDS; the tryptophan side chains pack against the backbone in DPC and point away from the backbone in SDS; the rms fluctuation of the peptide backbone and peptide side chains is greater in SDS than in DPC; and the peptide backbone order parameters are higher in DPC than in SDS.     

Cation-pi interaction in model alpha-helical peptides

Cation-pi interactions are increasingly recognized as important in chemistry and biology. Here we investigate the cation-pi interaction by determining its effect on the helicity of model peptides using a combination of CD and NMR spectroscopy. The data show that a single Trp/Arg interaction on the surface of a peptide can make a significant net favorable free energy contribution to helix stability if the two residues are positioned with appropriate spacing and orientation. The solvent-exposed Trp-->Arg (i, i + 4) interaction in helices can contribute -0.4 kcal/mol to the helix stability, while no free energy gain is detected if the two residues have the reversed orientation, Arg-->Trp (i, i + 4). The derived free energy is consistent with other experimental results studied in proteins or model peptides on cation-pi interactions. However in the same system the postulated Phe/Arg (i, i + 4) cation-pi interaction provides no net free energy to helix stability. Thus the Trp-->Arg interaction is stronger than Phe-->Arg. The cation-pi interactions are not sensitive to the screening effect by adding neutral salt as indicated by salt titration. Our results are in qualitative agreement with theoretical calculations emphasizing that cation-pi interactions can contribute significantly to protein stability with the order Trp > Phe. However, our and other experimental values are significantly smaller than estimates from theoretical calculations.     

Helical peptide models for protein glycation: proximity effects in catalysis of the Amadori rearrangement.

INTRODUCTION: Non-enzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. The observation that only a fraction of all lysine residues undergo glycation indicates the role of the immediate chemical environment in the glycation reaction. Here we have constructed helical peptide models, which juxtapose lysine with potentially catalytic residues in order to probe their roles in the individual steps of the glycation reaction. RESULTS: The peptides investigated in this study are constrained to adopt helical conformations allowing residues in the i and i+4 positions to come into spatial proximity, while residues i and i+2 are far apart. The placing of aspartic acid and histidine residues at interacting positions with lysine modulates the steps involved in early peptide glycation (reversible Schiff base formation and its subsequent irreversible conversion to a ketoamine product, the Amadori rearrangement). Proximal positioning of aspartic acid or histidine with respect to the reactive lysine residue retards initial Schiff base formation. On the contrary, aspartic acid promotes catalysis of the Amadori rearrangement. Presence of the strongly basic residue arginine proximate to lysine favorably affects the pK(a) of both the lysine epsilon-amino group and the singly glycated lysine, aiding in the formation of doubly glycated species. The Amadori product also formed carboxymethyl lysine, an advanced glycation endproduct (AGE), in a time-dependent manner. CONCLUSIONS: Stereochemically defined peptide scaffolds are convenient tools for studying near neighbor effects on the reactivity of functional amino acid sidechains. The present study utilizes stereochemically defined peptide helices to effectively demonstrate that aspartic acid is an efficient catalytic residue in the Amadori arrangement. The results emphasize the structural determinants of Schiff base and Amadori product formation in the final accumulation of glycated peptides.     

分子动力学研究F1-ATP合酶对三磷酸腺苷的稳定和定位作用

F₁-ATP合酶通过与ATP之间建立广泛的相互作用,实现对ATP的位置进行精确的定位.这些相互作用为ATP的合成/水解创造了稳定的环境.理解这些相互作用是理解ATP的合成/水解机理的基础.我们通过分子动力学模拟方法研究这些相互作用,找出在稳定化过程中起到重要作用的残基.通过检测ATP和F₁-ATP合酶之间的非键相互作用,发现残基段158-164所形成的loop区域及残基R189, Y345对ATP存在显著相互作用.其中,该loop区域对ATP的三磷酸部分形成一个半包围结构,封闭活性位点区域,并通过氢键网络约束ATP三磷酸的运动,为ATP合成/水解创造稳定的环境.此外,关键残基Y345通过π-π叠加相互作用对ATP的碱基进行约束,但是ATP的碱基可以在平行于Y345芳香环的平面内进行滑动,我们推断这种滑动运动有利于促进ATP的水解.     

Quantifying intermolecular interactions: guidelines for the molecular recognition toolbox

Molecular recognition events in solution are affected by many different factors that have hampered the development of an understanding of intermolecular interactions at a quantitative level. Our tendency is to partition these effects into discrete phenomenological fields that are classified, named, and divorced: aromatic interactions, cation-pi interactions, CH-O hydrogen bonds, short strong hydrogen bonds, and hydrophobic interactions to name a few.1 To progress in the field, we need to develop an integrated quantitative appreciation of the relative magnitudes of all of the different effects that might influence the molecular recognition behavior of a given system. In an effort to navigate undergraduates through the vast and sometimes contradictory literature on the subject, I have developed an approach that treats theoretical ideas and experimental observations about intermolecular interactions in the gas phase, the solid state, and solution from a single simplistic viewpoint. The key features are outlined here, and although many of the ideas will be familiar, the aim is to provide a semiquantitative thermodynamic ranking of these effects in solution at room temperature.     

Refined models for the preferential interactions of tryptophan with phosphocholines

A series of molecular models of the adducts formed between N-acetyl-l-tryptophan ethylamide and diacetyl-sn-glycero-3-phosphocholine have been generated. Using rOesy data that enabled us to place restrictions on the proximity of a number of key protons in the amino acid/phosphocholine pairs, a series of structures were generated following molecular dynamics and mechanics experiments using the CHARMM27 force field. These structures were then subjected to a series of clustering algorithms in order to classify the tight binding interactions between a single tryptophan and a phosphocholine. From these analyses, it is evident that: (i) binding is characterised by hydrogen bonding between the indole NH as donor and phosphate oxygen as acceptor, cation-carbonyl interactions between the choline ammonium and amide carbonyl groups and cation-pi interactions; (ii) cation-pi interactions are not always observed, particularly when their formation is at the expense of cation-carbonyl and hydrogen bonding interactions; (iii) on the basis of amino acid torsional parameters, it is possible to predict whether the phosphocholine headgroup will bind in a compact or elongated conformation. Extension of the procedures to characterise 2 : 1 Trp-PC binding revealed that the same intermolecular interactions are predominant; however, combinations of all three intermolecular interactions within the same adduct occur much more frequently due to the availability of donor/acceptor groups from both tryptophans in the 2 : 1 system.