The recognition of nucleotides with model beta-hairpin receptors: investigation of critical contacts and nucleotide selectivity

[reaction: see text] We have investigated the factors that contribute to binding of ATP by a designed 12-residue beta-hairpin peptide, WKWK, and have determined its selectivity for binding to the naturally occurring nucleotide triphosphates. We have previously shown that WKWK creates an ATP binding pocket on one face of the beta-hairpin consisting of two Trp and two Lys residues. Mutation of the two Lys residues on the binding face of the beta-hairpin resulted in a lower affinity, indicating that each is involved in ATP binding and that each residue contributes approximately -1.5 kcal/mol to the energy of complexation. Replacement of either Trp residue of the ATP binding pocket with Phe or Leu destabilizes the complex formed with ATP by approximately 1 kcal/mol, indicating that both Trp residues participate in interactions with ATP. For binding to the nucleotide triphosphates, the order of binding affinity was shown to follow dTTP > GTP > ATP > CTP, with differences in binding energies spanning as much as 1.6 kcal/mol. NMR analysis demonstrates that both aromatic interactions with the Trp side chains and CH-pi interactions between the ribose protons and the Trp residues may contribute significantly to binding. The results from our model system provide useful thermodynamic information regarding protein-nucleic acid interactions that occur at the surface of a beta-sheet.     

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.     

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.     

Analysis of the pi-pi stacking interactions between the aminoglycoside antibiotic kinase APH(3')-IIIa and its nucleotide ligands

A key contact in the active site of an aminoglycoside phosphotransferase enzyme (APH(3')-IIIa) is a pi-pi stacking interaction between Tyr42 and the adenine ring of bound nucleotides. We investigated the prevalence of similar Tyr-adenine contacts and found that many different protein systems employ Tyr residues in the recognition of the adenine ring. The geometry of these stacking interactions suggests that electrostatics play a role in the attraction between these aromatic systems. Kinetic and calorimetric experiments on wild-type and mutant forms of APH(3')-IIIa yielded further experimental evidence of the importance of electrostatics in the adenine binding region and suggested that the stacking interaction contributes approximately 2 kcal/mol of binding energy. This type of information concerning the forces that govern nucleotide binding in APH(3')-IIIa will facilitate inhibitor design strategies that target the nucleotide binding site of APH-type enzymes.     

Comparison of C-H...pi and hydrophobic interactions in a beta-hairpin peptide: impact on stability and specificity

We have examined the impact of C-H...pi and hydrophobic interactions in the diagonal position of a beta-hairpin peptide through comparison of the interaction of Phe, Trp, or Cha (cyclohexylalanine) with Lys or Nle (norleucine). NMR studies, including NOESY and chemical shift perturbation studies, of the Lys side chain indicates that Lys interacts in a specific geometry with Phe or Trp through the polarized C epsilon. In contrast, Nle does not interact in a specific manner with the diagonal aromatic residue. Thermal denaturation provides additional support that Lys and Nle interact in fundamentally different manners. Folding of the peptide with a diagonal Trp...Lys interaction was found to be enthalpically driven, whereas the peptide with a diagonal Trp...Nle interaction displayed cold denaturation, as did the control peptide with a diagonal Cha...Nle interaction, indicating different driving forces for interaction of Lys and Nle with Trp. These findings have significant implications for specificity in protein folding and de novo protein design.     

Multiple intermolecular interaction modes of positively charged residues with adenine in ATP-binding proteins

Adenosine 5'-triphosphate (ATP) plays an essential role in all forms of life. Molecular recognition of ATP in ATP-binding proteins is a subject of great importance for understanding enzymatic mechanisms and for drug design. We have carried out a large-scale data mining of the Protein Data Bank (PDB) to analyze molecular determinants for recognition of ATP, in particular, the adenine base, by ATP-binding proteins. A novel distribution pattern of charged residues around the adenine base was discovered: lysine residues tend to occupy the major groove N7 side of the adenine base, and the arginine residues situate preferentially above or below the adenine bases. Such an arrangement is advantageous because it facilitates multiple modes of intermolecular interactions, that is, cation-pi interactions and a hydrogen bond between lysine and adenine, and cation-pi and pi-pi stacking interactions between arginine and adenine. For the two representative Lys...Adenine and Arg...Adenine interactions, intermolecular interaction energies were subsequently analyzed by means of the supermolecular approach at the MP2 level with solvation free energy correction using the SM5.42R model of Cramer and Truhlar, which gave rise to significant interaction strengths.     

Force-induced insulin dimer dissociation: a molecular dynamics study

Understanding the forces and dynamics of insulin dissociation is critical for devising formulations for the treatment of insulin-dependent diabetes. In earlier work, we applied AFM-based force spectroscopy to covalently tethered and oriented insulin monomers to assess the effect of molecular orientation on insulin-insulin binding forces. We report here on steered molecular dynamics simulations of the insulin dissociation force spectroscopy experiment. Consistent with our experiments, our simulation results suggest that insulin dimer dissociation occurs near the limit of extensibility of the B-chain. We have also found that the forced dissociation of the insulin dimer is a rate-dependent process, involving significant conformational changes to the monomer(s). The insulin dimer dissociation pathway also depends on the relative strength of the inter-monomer interactions across the antiparallel beta-sheet interface and the intra-monomer interactions of residues A1 and A30 with the insulin B-chain. Our simulation results strongly support the design of bioactive insulin analogues that involves altering hydrogen bonding and hydrophobic interactions across the beta-sheet dimer interface.     

Minimization and optimization of designed beta-hairpin folds

Minimized beta hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, DeltaG(U)(298) >or= +7 kJ/mol) and 12-residue (KTWNPATGKWTE, DeltaG(U)(298) = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-pi interaction between Lys(1) and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hepsilon3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the -TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small DeltaC(p) for unfolding (+420 J K(-)(1)/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH(2)) with the fold stability reduced to DeltaG(U)(278) = -0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also form the same hairpin-stabilizing W/W interaction.     

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.     

Turn residues in beta-hairpin peptides as points for covalent modification

Beta-turns are important sites for protein-protein and protein-peptide interactions, but little research has explored synthetic modifications of turn residue side-chains in a beta-hairpin peptide. To this end, beta-hairpin peptides were synthesized containing the type I' turn sequence Val-Asn-Gly-Lys with modifications at Asn and Lys. We found that these variations impose a small penalty, demonstrating that beta-turns are capable of displaying a range of functionality, which may be exploited for biomolecular recognition and medicinal applications. [structure: see text]     

A small aptamer with strong and specific recognition of the triphosphate of ATP

We report the in vitro selection of an RNA-based ATP aptamer with the ability to discriminate between adenosine ligands based on their 5' phosphorylation state. Previous selection of ATP aptamers yielded molecules that do not significantly discriminate between ligands at the 5' position. By applying a selective pressure that demands recognition of the 5' triphosphate, we obtained an aptamer that binds to ATP with a Kd of approximately 5 muM, and to AMP with a Kd of approximately 5.5 mM, a difference of 1100-fold. This aptamer demonstrates the ability of small RNAs to interact with negatively charged moieties.     

Influence of a terminal formamido group on the sequence recognition of DNA by polyamides

Pyrrole (Py)-imidazole (Im)-containing polyamides bind in the minor groove of DNA and can recognize specific sequences through a stacked antiparallel dimer. It has been proposed that there are two different low energy ways to form the stacked dimer and that these are sensitive to the presence of a terminal formamido group: (i) a fully overlapped stacking mode in which the N-terminal heterocycles of the dimer stack on the amide groups between the two heterocycles at the C-terminal and (ii) a staggered stacking mode in which the N-terminal heterocycles are shifted by approximately one unit in the C-terminal direction (Structure 1997, 5, 1033-1046). Two different DNA sequences will be recognized by the same polyamide stacked in these two different modes. Despite the importance of polyamides as sequence specific DNA recognition agents, these stacking possibilities have not been systematically explored. As part of a program to develop agents that can recognize mismatched base pairs in DNA, a set of four polyamide trimers with and without terminal formamido groups was synthesized, and their interactions with predicted DNA recognition sequences in the two different stacking modes were evaluated. Experimental difficulties in monitoring DNA complex formation with polyamides were overcome by using surface plasmon resonance (SPR) detection of the binding to immobilized DNA hairpin duplexes. Both equilibrium and kinetic results from SPR show that a terminal formamido group has a pronounced effect on the affinity, sequence specificity, and rates of DNA-dimer complex formation. The formamido polyamides bind preferentially in the staggered stacking mode, while the unsubstituted analogues bind in the overlapped mode. Affinities for cognate DNA sequences increase by a factor of around 100 when a terminal formamido is added to a polyamide, and the preferred sequences recognized are also different. Both the association and the dissociation rates are slower for the formamido derivatives, but the effect is larger for the dissociation kinetics. The formamido group thus strongly affects the interaction of polyamides with DNA and changes the preferred DNA sequences that are recognized by a specific polyamide stacked dimer.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.     

NMR structure of a cyclic polyamide-DNA complex

The solution structure of a cyclic polyamide ligand complexed to a DNA oligomer, derived from NMR restrained molecular mechanics, is presented. The polyamide, cyclo-gamma-ImPyPy-gamma-PyPyPy-, binds to target DNA with a nanomolar dissociation constant as characterized by quantitative footprinting previously reported. 2D (1)H NMR data were used to generate distance restraints defining the structure of this cyclic polyamide with the DNA duplex d(5'-GCCTGTTAGCG-3'):d(5'-CGCTAACAGGC-3'). Data interpretation used complete relaxation matrix analysis of the NOESY cross-peak intensities with the program MARDIGRAS. The NMR-based distance restraints (276 total) were applied in restrained molecular dynamics calculations using a solvent model, yielding structures with an rmsd for the ligand and binding site of approximately 1 A. The resulting structures indicate some distortion of the DNA in the binding site. The constraints from cyclization lead to altered stacking of the rings in the halves of the cyclic ligand relative to unlinked complexes. Despite this, the interactions with DNA are very similar to what has been found in unlinked complexes. Measurements of ligand amide and DNA imino proton exchange rates indicate very slow dissociation of the ligand and show that the DNA can undergo opening fluctuations while the ligand is bound although the presence of the ligand decreases their frequency relative to the free DNA.     

Controlled aggregation of adenine by sugars: physicochemical studies, molecular modelling simulations of sugar-aromatic CH-pi stacking interactions, and biological significance

CH-Pi stacking interactions between carbohydrates and aromatic compounds play a central role in biomolecular recognition, especially in lectin-sugar and protein-glycolipid systems. In the present study, we have measured the solubility of the sparingly soluble aromatic base adenine in presence of various saccharides as an approach to investigate the interaction between adenine and sugars. Above 82.5 mM, adenine solutions gradually formed a crystalline precipitate which could be quantified by spectrophotometric turbidity measurements. Precipitation of adenine was increased by salts (NaCl and NaF) whereas it was prevented by DMSO, in agreement with the involvement of hydrophobic interactions (pi-pi stacking) in the vertical stacking of adenine molecules. Several monosaccharides and disaccharides were found to increase adenine solubility, with the following order: D-galactose = D-lactose > D-sucrose > D-glucose = D-maltose > D-ribose > D-fructose. Molecular mechanics simulations indicated that the potent cosolvent effect of beta-D-galactopyranose was probably mediated by CH-pi stacking interactions between its apolar surface and the aromatic structure of adenine. The polar OH groups of the sugars interacted with surrounding water molecules, ensuring the solubility of sugar-adenine complexes. In contrast, beta-D-fructofuranose, which has two polar faces, did not stack onto adenine and had a weak cosolvent effect. CH-pi stacking interactions were also demonstrated between 6-methylpurine and the sugar head group of glycolipids (glucosyl-, galactosyl- and lactosylceramide) but not with the charged head group of phosphatidylinositol-4,5-diphosphate. These data indicate that galactose-containing molecules have a high stacking propensity for aromatic compounds such as adenine, due to the specific structure of the galactose cycle.     

Design and construction of an open multistranded beta-sheet polypeptide stabilized by a disulfide bridge

The design and characterization of an open eight-stranded beta-sheet in a synthetic, 2-fold symmetric 70-residue peptide is described. The design strategy involves the generation of a 35-residue four-stranded beta-sheet peptide in which successive hairpins are nucleated by appropriately positioned (D)Pro-Xxx sequences. Oxidative dimerization using a single Cys residue positioned at the center of the C-terminal strand results in a disulfide-bridged eight-stranded structure. Nuclear Overhauser effects firmly establish an eight-stranded beta-sheet in methanol. In water, the outer strands are frayed, but a well-defined four-stranded beta-sheet stabilized by a disulfide bridge and a hydrophobic cluster is determined from NMR data. Comparison of the precursor peptide with the disulfide-bridged dimer reveals considerable enhancement of beta-sheet content in the latter, suggesting that the disulfide cross-link is an effective strategy for the stabilization of beta-sheets.     

Self-assembly of peptide porphyrin complexes: toward the development of smart biomaterials

The anionic porphyrin, meso-tetrakis(4-sulfonatophenyl)porphine, is found to tightly bind to an engineered 14-residue peptide, resulting in induced alpha-helix formation when mixed in aqueous solutions. The small porphyrin-peptide dissociation constant (2 muM) observed is related to the energetics of peptide helix formation coupled with electrostatic interactions between the anionic porphyrin and cationic residues in the coiled peptide. Analytical ultracentrifugation measurements indicate the porphyrin-peptide complexes dimerize, probably into a coiled coil, and weakly associate to form even higher order structures.     

Tryptophan side chain electrostatic interactions determine edge-to-face vs parallel-displaced tryptophan side chain geometries in the designed beta-hairpin "trpzip2"

The interaction geometries of the four tryptophan (Trp) side chains in the 12-residue designed beta-hairpin trpzip2 are investigated using all-atom explicit-solvent molecular dynamics simulations. The experimentally observed edge-to-face (EtF) pairwise interaction geometries are stable on a time scale of 10 ns. However, removing the electrostatic multipoles of the Trp side chains while retaining the dipoles of the side chains' NH moieties induces a conformational change to a geometry in which three of the four side chains interact in a parallel-displaced (PD) manner. Free energy simulations of the Etf to PD conformational change reveal that, with the side chain multipole moments intact (+MP), the EtF conformation is preferred by 5.79 kcal/mol. Conversely, with only the dipole moments of the side chain NH moieties intact (-MP), the PD conformation's free energy is more favorable by 1.71 kcal/mol. In contrast to energetic similarities for Trp side chain-water electrostatic and Trp side chain-Trp side chain and Trp side chain-water van der Waals, +MP Trp side chain-Trp side chain electrostatic interactions are more favorable by 4.21 kcal/mol in the EtF conformation, while in the -MP case the EtF and PD conformations' Trp side chain-Trp side chain electrostatic energies are nearly identical. The results highlight the importance of electrostatic multipole moments in determining aromatic-aromatic interaction geometries in aqueous biomolecular systems and argue for the inclusion of this physics in simplified models used for protein-ligand docking and protein structure prediction, possibly through a truncated Coulomb term between aromatic moieties.     

Thermodynamics and the mechanism of interaction of adenine with amino acids in water

Two groups of amino acids, which react differently with adenine, are distinguished. In the case of nonpolar and aliphatic amino acids, the endothermic effect of dehydration plays a decisive role, while in the case of aromatic, polar, and charged amino acids the exothermic effect of interaction with adenine is dominant. Associates of Ade with Lys. HCl, His, Trp, Asp, and Glu were found. It was demonstrated that the complex-forming ability of purines (Ade and Caf) is higher than that of pyrimidines. Based on the linear enthalpy-entropy compensation effect for complexes of amino acids with adenine, it was suggested that the hydration state of interacting molecules contributes significantly to interactions of Ade with amino acids.