Theoretical studies on protein-nucleic acid interactions. III. Stacking of aromatic amino acids with bases and base pairs of nucleic acids

Stacking of aromatic amino acids tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), and histidine (His) with bases and base pairs of nucleic acids has been studied. Stacking energies of the amino acid–base (or base pair) complexes have been calculated by second-order perturbation theory. Our results show that, in general, the predominant contribution to the total stacking energy comes from the dispersion terms. In these cases, repulsion energy is greater than the sum of electrostatic and polarization energies. In contrast to this, interaction of histidine with the bases and base pairs is largely Coulombic in nature. The complexes of guanine with aromatic amino acids are more stable than the corresponding complexes of adenine. Among pyrimidines, cytosine forms the most stable complexes with the aromatic amino acids. The G · C base pair has the highest affinity with aromatic amino acids among various sets of base pairs. Optimized geometries of the stacked complexes show that the aromatic moieties overlap only partially. The heteroatom of one residue generally overlaps with the other aromatic moiety. There is a considerable degree of configurational freedom in the stacked geometries. The role of stacking in specific recognition of base sequences by proteins is discussed.     

Several properties of complexes of polynucleotides with aromatic amino acid amides of oligonucleotides

Residues of Phe, Tyr and Trp in the complexes of their oligonucleotide amidates and polynucleotides of A-U of G-C nucleotide composition are most likely localized in the minor groove of the Watson--Crick part of the triple helix where they interact with bases but do not intercalate into the helix. Formation of the complexes is accompanied with a change in the relative localization of amino acids and bases. The major geometrical parameters of the triple helices of the complexes are not changed by the residues of aromatic amino acids (according to CD data). A slight violation of stacking interactions between bases is observed along with an increase of the cooperativity of melting of the complexes of A-U composition (according to UV absorption data). The effect of the residues of aromatic amino acids on the stability of triple helices is determined by the nucleotide composition of the latter, i.e. complexes of A-U composition are destabilized with the Phe, Tyr and Trp residues, whereas the Trp residue does not affect the stability of the complexes of G-C composition. The hydrophobic character of aromatic amino acids and their different affinity for bases of different structure seem to account for this difference in stability. The dependence of the thermal stability of RNH-dp(An).2poly(U)-complexes on the structure of the amide radical (residues of glycin, aromatic amino acids, alkyl- and arylalkyl amines) testifies the ability of the radical to "regulate" the interaction between the oligomer and the complementary polynucleotide. This capacity for "regulation" is not observed in the system of G-C composition.     

Effect of water coordination on competition between π and non-π cation binding sites in aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan Li+, Na+, and K+ complexes

Quantum chemistry methods have been applied to charged complexes of the alkali metals Li(+), Na(+), and K(+) with the aromatic amino acids (AAAs) phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). The geometries of 72 different complexes (Phe·M, Tyr·M, Trp·M, M is Li(+), Na(+), or K(+)) were completely optimized at the B3LYP/6-311+G(d,p) level of density functional theory. The solvent effect on the geometry and stability of individual complexes was studied by making use of a microsolvation model. The interaction enthalpies, entropies, and Gibbs energies of nine different complexes of the systems Phe·M, Tyr·M, and Trp·M (M is Li(+), Na(+), or K(+)) were also determined at the B3LYP density functional level of theory. The calculated Gibbs binding energies of the M(+)-AAA complexes follow the order Phe < Tyr < Trp for all three metal cations studied. Among the three AAAs studied, the indole ring of Trp is the best π donor for alkali metal cations. Our calculations demonstrated the existence of strong cation-π interactions between the alkali metals and the aromatic side chains of the three AAAs. These AAAs comprise about 8% of all known protein sequences. Thus, besides the potential for hydrogen-bond interaction, aromatic residues of Phe, Tyr, and Trp show great potential for π-donor interactions. The existence of cation-π interaction in proteins has also been demonstrated experimentally. However, more complex experimental studies of metal cation-π interaction in diverse biological systems will no doubt lead to more exact validation of these investigations.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.     

Functional and structural consequences of aromatic residue substitutions within the kringle-2 domain of tissue-type plasminogen activator.

Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp25 is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2tpA), whereas Trp63, Trp74, and Tyr76 function in omega-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2tPA, viz., Tyr2, Phe3, Tyr9, Tyr35, Tyr52, have been subjected to structure-function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr35 destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting temperatures, changes at Tyr9 and Tyr52 generally destabilized the native structure of K2tPA to a greater extent than changes at Tyr2, Phe3, and Tyr35. Taken together, results to date show that, in concert with predictions from the crystal structure of K2tpA, ligand binding appears to rely most on the integrity of Trp63 and Trp74, and aromaticity at Tyr76. With regard to aromatic amino acids, kringle folding is most dependent on Tyr9, Trp25, Tyr52, Trp63, and Tyr76. As yet, no obvious major roles have been uncovered for Tyr2, Phe3, or Tyr35 in K2tpA.     

Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.     

Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY

BackgroundThe structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals.MethodsThe 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra.ResultsThe MSG proteins, selectively labeled with either [δ₁,ε₁,ε₃,η₂]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously.ConclusionsWe demonstrated that the aromatic ring ¹³CH pairs without directly bonded ¹³C and adjacent ¹H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available.General significanceThe method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.     

Optimization of aromatic side chain size complementarity in the hydrophobic core of a designed coiled-coil.

The coiled-coil structure plays an important roles, especially in protein assembly. Previously we constructed AAB-type heterotrimeric coiled-coils by manipulating the packing in the hydrophobic core using Trp and Ala residues, where one Trp and two Ala residues were placed in the hydrophobic core instead of three Ile residues. To optimize the packing complementarity in the hydrophobic core, we investigated the effects of introducing various aromatic amino acids on the formation of an AAB-type heterotrimeric coiled-coil, by circular dichroism, thermal stability, and nuclear magnetic resonance (NMR) studies. We found that the Phe residue was more suitable for heterotrimeric coiled-coil formation than the Trp residue, when combined with two Ala residues, whereas the Tyr and His residues did not induce the coiled-coil structure efficiently.     

Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts

Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.     

π–π Interactions in Structural Stability: Role in RNA Binding Proteins

RNA binding proteins play significant roles in many bio-macromolecular systems. Aromatic amino acid residues are vital for several biological functions. In the present work, the influences of π–π interactions in RNA binding proteins are analyzed. There are a total of 3,396 π-residues in RNA binding proteins out of which 1,547, 1,241, and 608 are phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), respectively. Among these 945, 634, and 356 Phe, Tyr, and Trp residues, respectively, are involved in π–π interactions. The observations indicate that majority of the aromatic residues in RNA binding proteins are involved in π–π interactions. Side chain–side chain π–π interactions are the predominant type of interactions in RNA binding proteins. These π–π interactions stabilize the core regions within RNA binding proteins. π–π interacting residues are evolutionary conserved. Residue-wise analysis indicates that π–π interacting residues have higher long-range contacts and hence they are important in the global conformational stability of these proteins.     

Dynamic cross-talk among remote binding sites: the molecular basis for unusual synergistic allostery

Allosteric regulation of protein function is critical for metabolic control. Binding of allosteric effectors elicits a functional change in a remote ligand binding site on a protein by altering the equilibrium between different forms in the protein ensemble. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids Trp, Phe, and Tyr. Feedback regulation by the aromatic amino acids is important for controlling the cellular levels of the aromatic amino acids, and many organisms have two or more DAH7PS isozymes that show differing sensitivities to aromatic compounds. Mycobacterium tuberculosis expresses a single DAH7PS that is insensitive to the presence of a single amino acid yet shows extraordinary synergistic inhibition by combinations of the pathway end products Trp and Phe. The Trp+Phe-bound structure for M. tuberculosis DAH7PS, showing two separate binding sites occupied by Trp and Phe for each monomer of the tetrameric protein, was obtained by cocrystallization. Comparison of this structure with the ligand-free M. tuberculosis DAH7PS demonstrates that there is no significant change in conformation upon ligand binding, suggesting that contributions from altered dynamic properties of the enzyme may account for the allosteric inhibition. Isothermal titration calorimetry experiments demonstrate that the inhibitor binding sites are in direct communication. Molecular dynamics simulations reveal different changes in dynamic fluctuations upon single ligand binding compared to dual ligand binding. These changes account for the cross-talk between inhibitor binding sites and the active site, simultaneously potentiating both dual ligand binding and diminution of catalytic function.     

Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9–2.6 Å. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.     

Histidine-aromatic interactions in barnase. Elevation of histidine pKa and contribution to protein stability.

Aromatic side-chains are found in the vicinity of histidine residues in many proteins and protein complexes. We have studied the interaction between a histidine residue (His18) and aromatic residues at position 94 in barnase. Three different techniques have been applied to show that Trp94 interacts more strongly with the protonated form of His18. The aromatic-histidine interaction stabilizes the protonated form of histidine by 0.8 to 1 kcal mol-1 relative to the unprotonated and, thereby, increases its pKa value. This was shown indirectly from the pH dependence of the stability of the wild-type protein and the mutant Trp94----Leu; and directly from the difference in pKa of His18 between wild-type barnase and the same mutant protein, and from double-mutant cycles that measure the total interaction energy of Trp94 with His18 at both low and high pH. When Trp94 is replaced by other aromatic amino acids, the strength of the interaction decreases in the series His-Trp greater than His-Tyr greater than His-Phe. The interaction is not masked by high salt concentrations. The raising of the pKa value of His18 by interaction with Trp94 is shown to be consistent with solution studies with model compounds. The histidine-aromatic interaction could have implications in binding and catalysis for modulation of the histidine pKa value.     

Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1-150) and the C terminus of its cysteine-rich domain (amino acids 190-300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692-702 from the C terminus of the alpha subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteine-rich domain and amino acids 692-702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of Asp(8), Asn(11), Tyr(28), His(30), Leu(33), Leu(56), Phe(58), Arg(59), and Trp(79) produced a 2- to 10-fold decrease in affinity and alanine mutation of Phe(90) resulted in a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of Arg(240), Phe(241), Glu(242), and Phe(251) produced a 2- to 10-fold decrease in affinity. In the region between amino acids 692 and 702, alanine mutation of Phe(701) produced a receptor devoid of binding activity and alanine mutations of Phe(693), Glu(693), Asn(694), Leu(696), His(697), Asn(698), and Ile(700) exhibited decreases in affinity ranging from 10- to 30-fold. With the exception of Trp(79), the disruptive mutants in L1 form a discrete epitope on the surface of the receptor. Those in the cysteine-rich domain essential for intact affinity also form a discrete epitope together with Trp(79).     

Arc repressor-operator DNA interactions and contribution of Phe10 to binding specificity

Solution properties of Arc repressors (wild-type and F10H variant) from Salmonella bacteriophage P22 and their complexes with operator DNA (Arc-wt-DNA and Arc-F10H-DNA) were characterized by circular dichroism, fluorescence, and Raman difference spectroscopy and compared with the crystal structures of free and DNA-bound Arc repressors (wild-type and F10V variant). From the crystal structure of Arc-wt-operator DNA complex, it is known that amino acids Phe10/10' flip out of the hydrophobic protein core, and in the Arc-F10V-DNA complex, the methyl groups of Val10/10' rotate toward the DNA. Arc-wt and Arc-F10H significantly perturb the Raman signatures of the operator DNA upon complex formation. The two proteins induce similar changes in the DNA spectra. Raman markers in the difference spectra (spectrum of the complex minus spectra of DNA and Arc) indicate binding of Arc in the major groove, several direct contacts, e.g., hydrogen bonds of protein residues with bases, and slight perturbations of the deoxyribose ring systems that are consistent with bending of the operator DNA. Trp14, the only one tryptophan of Arc repressor monomers, serves as a very sensitive tool for changes of the hydrophobic core of the protein. The Raman spectra identify in the free Arc-F10H variant a largely different chi(2,1) rotation angle of Trp14 compared to that in wild-type Arc. In the Arc-wt-DNA and Arc-F10H-DNA complexes, however, the Trp14 chi(2,1) rotation angles are similar in both proteins. Furthermore, in both complexes, a strengthening of the van der Waals interactions of the aromatic ring of Trp14 is indicated compared to these interactions in the free proteins. According to the fluorescence and Raman data, His10 is buried in the hydrophobic core of free Arc-F10H, resembling the "core" conformation of Phe10 in Arc-wt, but His10 is looped out in the complex with DNA resembling the "bound" conformation of Phe10 in the Arc-wt-operator DNA complex.     

Characterization of the nucleic acid-binding activities of the isolated amino-terminal head domain of the intermediate filament protein vimentin reveals its close relationship to the DNA-binding regions of some prokaryotic single-stranded DNA-binding proteins.

In order to demonstrate that the nucleic acid-binding activities of vimentin are dictated by its Arg-rich N-terminal head domain, this was cut off at position Lys96 with lysine-specific endoproteinase and analysed for its capacity to associate with a variety of synthetic and naturally occurring nucleic acids. The isolated polypeptide (vim NT) showed a preference for single-stranded (ss) polynucleotides, particularly for ssDNAs of high G-content. A comparison of the sequence and predicted secondary structure of vim NT with that of two prokaryotic ssDNA-binding proteins, G5P and G32P of bacteriophages fd and T4, respectively, revealed that the nucleic acid-binding region of all three polypeptides is almost entirely in the beta-conformation and characterized by a very similar distribution of aromatic amino acid residues. A partial sequence of vim NT can be folded into the same beta-loop structure as the DNA-binding wing of G5P of bacteriophage fd and related viruses. As in the case of G5P, nitration of the Tyr residues with tetranitromethane was blocked by single-stranded nucleic acids. This and spectroscopic data indicate intercalation of the Tyr aromatic ring systems between the bases of the nucleic acids and thus the contribution of a stacking component to the binding reaction. The binding was accompanied by significant changes in the ultraviolet absorption spectra of both vim NT and single-stranded nucleic acids. Upon mixing of vim NT with nucleic acids, massive precipitation of the reactants occurred, followed by the quick rearrangement of the aggregates with the formation of specific and soluble association products. Even at very high ionic strengths, at which no electrostatic reaction should be expected, a distinct fraction of vim NT incorporated naturally occurring ssRNAs and ssDNAs into fast sedimenting complexes, suggesting co-operative interaction of the polypeptide with the nucleic acids. In electron microscopy, the complexes obtained from 28 S rRNA appeared as networks of extended nucleic acid strands densely covered with vim NT, in contrast to the compact random coils of uncomplexed RNA. The networks produced from fd DNA were heterogeneous in appearance and their nucleoprotein strands in rare cases were very similar to the rod-like structures of G5P-fd DNA complexes.