The multiple roles of histidine in protein interactions
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Among the 20 natural amino acids histidine is the most active and versatile member that plays the multiple roles in protein interactions, often the key residue in enzyme catalytic reactions. A theoretical and comprehensive study on the structural features and interaction properties of histidine is certainly helpful.
Four interaction types of histidine are quantitatively calculated, including: (1) Cation-π interactions, in which the histidine acts as the aromatic π-motif in neutral form (His), or plays the cation role in protonated form (His+); (2) π-π stacking interactions between histidine and other aromatic amino acids; (3) Hydrogen-π interactions between histidine and other aromatic amino acids; (4) Coordinate interactions between histidine and metallic cations. The energies of π-π stacking interactions and hydrogen-π interactions are calculated using CCSD/6-31+G(d,p). The energies of cation-π interactions and coordinate interactions are calculated using B3LYP/6-31+G(d,p) method and adjusted by empirical method for dispersion energy.
The coordinate interactions between histidine and metallic cations are the strongest one acting in broad range, followed by the cation-π, hydrogen-π, and π-π stacking interactions. When the histidine is in neutral form, the cation-π interactions are attractive; when it is protonated (His+), the interactions turn to repulsive. The two protonation forms (and pKa values) of histidine are reversibly switched by the attractive and repulsive cation-π interactions. In proteins the π-π stacking interaction between neutral histidine and aromatic amino acids (Phe, Tyr, Trp) are in the range from -3.0 to -4.0 kcal/mol, significantly larger than the van der Waals energies.
KeywordsAmino acids Histidine Protonation Protein interaction Protein structure
The molecular interactions of histidine with other amino acids and metallic cations in proteins can be classified into the following five types. (1) Cation-π interaction[7, 8, 9]. The side chain imidazole of His is an aromatic ring. Histidine can take part in the cation-π interactions as the aromatic motif with metallic cations or organic cations (protonated amino acids, Lys+ and Arg+) [7, 9, 10, 11]. On the other hand, the protonated His+ is an organic cation, which can join the cation-π interactions as an organic cation with other aromatic amino acids (Phe, Tyr, and Trp) [12, 13, 14, 15, 16]. (2) π-π stacking interaction[17, 18, 19, 20]. The imidazole structure of histidine side chain is a conjugative π-plane, which can make π-π stacking interactions with the aromatic side chains of other amino acids (Phe, Tyr, and Trp) [20, 21]. (3) hydrogen-π interaction[22, 23]. The polar hydrogen atom of histidine can form hydrogen-π bond with other aromatic amino acids in ‘T’ orientation. (4) Coordinate bond interaction[3, 24, 25]. The basic nitrogen atom in the imidazole of histidine has a lone electron pair that make it a coordinate ligand of metallic cations, such as Zn2+ and Ca2+[26, 27]. (5) Hydrogen bond interaction[28, 29, 30, 31]. The polar hydrogen atom of the imidazole is a hydrogen-bond donor, and the basic nitrogen atom is a hydrogen-bond acceptor.
In protein interactions the roles of histidine are complicated by the five interaction types and two protonation forms. The unique behaviors of histidine have been discussed in literatures from different aspects [7, 32]. However, the quantitative interaction energies of five interaction types and the factors affecting the interaction energies still need more investigations. The influences of five interaction types to the protonation form (and pKa value) of histidine are still unclear. In this study the multiple roles of histidine in molecular interactions are quantitatively studied using quantum chemical calculations, and the factors, which influence the interaction energies and pKa value of histidine in proteins, are analyzed in detail.
Methods and materials
Comparison of three methods (DFT, CCSD, and CCSD(T)) for five interaction types (cation-π; π-π staking; hydrogen-π; hydrogen bond; and metallic cation-coordinate interaction
a π-π stack
From the data in Table 1 we find that the DFT method B3LYP cannot yield attractive interaction energy in C6H6-C6H6 π-π stacking interaction, completely failing in describing the π-π stacking interactions, which are dispersion dominated phenomenon. On the other hand the higher level method CCSD calculation produces attractive C6H6-C6H6 π-π stacking energy −1.883 kcal/mol. In this study the energy differences between B3LYP and CCSD are used as the dispersion contribution in the molecular interaction energies. In the hydrogen-π interaction more than 50% interaction energy is from the dispersion contribution. The interaction energies of other three interaction types (cation-π interactions, common hydrogen bond interactions, and metal cation-His coordinate interactions), obtained by using B3LYP and CCSD methods, have no remarkable difference. In above three interaction types the electrostatic (charge) interactions and orbital coordinate interactions make the main contributions, and the contribution of dispersion interactions are less than 10% [8, 41, 42]. In the C6H6CH3-H3O+ cation-π calculations the CPU time of three methods (B3LYP, CCSD, and CCSD(T)) are 1.08 hours, 50 days, and 86 days, respectively. However, the energy difference of cation-π interaction between B3LYP and CCSD(T) is only 1.08 kcal/mol, less than 6%.
In this study the π-π stacking interactions and the hydrogen-π interactions are calculated using CCSD/6-31+G(d,p) method, and the B3LYP/6-31+G(d,p) is used in the calculations of cation-π interactions and ligand-cation coordinate interactions. In recent years great efforts are made to make up the shortcoming of DFT in dispersion interactions, including design of new functional , or empirical correction terms [41, 42, 48, 49, 50]. , In this study the missing dispersion energies in DFT calculations are corrected by an empirical method suggested by Du et al . The interaction energies in solutions are calculated by using the polarizable continuum model (PCM) [50, 51, 52, 53].
In this study most molecule monomers are optimized by using CCSD/6-31+G(d,p) methods. Some large amino acids, such as Tyr and Trp, first are optimized at B3LYP/6-31+G(d,p) level, then the side chains are optimized at CCSD/6-31+G(d,p) level. The geometry parameters of side-chain, obtained from CCSD calculations, are combined with the parameters of DFT optimizations. In this study the protonated His+ is simplified as the protonated imidazole (C3N2H4+), protonated Arg+ is simplified as CHNH2NH2+, and the protonated amino acid Lys+ is simplified as CH3NH3+, respectively, as shown in Figure 1C, D, and E. The structures of four interaction types (cation-π interaction, π-π stacking interaction, hydrogen-π interaction, and coordinate bond interaction) of His are shown in Figure 1F, G, H, and I, respectively. Usually amino acids have several stable structural conformations with different energies. In proteins the orientations of residue side chains and the structural conformations of peptide backbone are innumerous. The optimized structures of amino acids, shown in Figure 1, are only one of the possible conformations. In Figure 1 F the metallic cation can be put at the upside or at the downside of the aromatic planes. In the ‘Upside’ structure the cation-π interaction may be complicated by the interaction elements in peptide backbone. On the other hand, the ‘Downside’ structure is less affected by other interaction elements. In this study we focus on the ‘pure’ cation-π interactions, the ‘Downside’ structures. All calculations are performed on Sugon-5000A computer using Gaussian 09 software package . The detailed geometrical parameters of optimized molecular structures are stored in supporting material (Optimized-Mol.zip).
In this section all calculation results are reported and summarized using tables and figures. Brief comparisons and illustrations are provided following the calculation results. Four interaction types (cation-π, π-π stacking, hydrogen-π, and coordinate bond interaction) of histidine with other amino acids and metallic cations are calculated in gas phase and in solutions (water, acetonitrile, and cyclohexane). The hydrogen bonding interaction of histidine is not included in this study, because it is a familiar and well studied interaction type.
Cation-π interactions of Histidine
Cation-π interaction energies between amino acid His and cations in gas phase
His (Aromatic motif)
Cation-π interaction energies between histidine (His) and cations in three solvents (water, acetonitrile, and cyclohexane)
π-π stacking interactions of histidine
The π-π stacking interaction energies between His and aromatic amino acids in gas phase
The π-π stacking energies increase with the size of π-system. In Table 4 the π-π stacking energy (−4.035 kcal/mol) of His-Trp is larger than that of His-Phe and His-Tyr because of the larger π-system of Trp. In DNA the π-π stacking interactions have larger contributions than in proteins [41, 47, 48, 49]. The protonated amino group (CHNH2NH2+) of Arg+ forms a π-plane, and the larger π-π stacking energy (−5.0432 kcal/mol) of His-Arg+ may partially from the cation-π interaction. The lower part of Table 4 lists the π-π stacking interaction energies between the protonated His+ and three aromatic amino acids (Phe, Tyr, and Trp), which are remarkably larger than that in the up part of Table 4.
Hydrogen-π interactions of histidine
The hydrogen-π interaction energies between His and aromatic amino acids in gas phase
Comparing the data in Table 4 and Table 5, the energies of hydrogen-π interactions are larger than the corresponding energies of π-π stacking interactions. In proteins the energies of hydrogen-π interactions are in the range −5 to −8 kcal/mol, comparable to the common hydrogen bond interactions (−4 to −6 kcal/mol). Actually, the π-π stacking interaction energies of aromatic amino acids contain the contributions of hydrogen-π interactions from the polar hydrogen atoms in His and in Tyr.
Coordinate bonding interactions between His and cations
The coordinate bonding interaction energies between His and metallic ations in gas phase and in solutions
Water (ε =78.39)
Histidine is an ionizable amino acid with the acidic ionization constant around pKa=6.5, very close to neutral. An interesting finding in this study is that the protonation of histidine has closely relationship with the interaction types. The cation-π interactions of neutral histidine (His) are attractive, and the cation-π interactions of protonated histidine (His+) are repulsive. A reasonable deduction is that pH condition can reversibly switch the cation-π interactions of histidine from attractive to repulsive. Vice versa, the cation-π interactions can affect the two protonation forms of histidine. In proteins the pKa value of His can change in a broad range due to the influence of interaction environment, and histidine can play the roles of both proton donor or acceptor [58, 65, 66]. The stronger attractive cation-π interaction can make the pKa value of His lower, and the lower pH condition may turn the cation-π interaction from attractive to repulsive. For the same reason, other interaction types (coordinate interaction, hydrogen-π interaction, hydrogen bond and the π-π stacking interaction) may also affect the pKa value of histidine to some degree .
In protein hydrolysis reactions the pKa value of His is a critically important property. In the catalytic triads of lipase, the basic nitrogen of histidine is used to abstract a proton from threonine, serine, or cysteine to activate it as a nucleophile. In carbonic anhydrases, a histidine proton shuttle is utilized to rapidly transport protons away from a zinc-bound water molecule to quickly regenerate the active form of the enzyme [67, 68]. In the histidine proton shuttle, histidine abstracts a proton with its basic nitrogen to make a positively-charged intermediate, and then use another molecule, a buffer, to extract the proton from its acidic nitrogen. Our study illustrates that in the proton shuttle procedure, the histidine is not working by itself alone, but with the collaboration of environmental residues through the multiple interactions that affect the pKa value of histidine.
Based on our calculation results the energy order of five interaction types (cation-π interaction, π-π stacking interaction, hydrogen-π interaction, hydrogen-bond interaction, and coordinate bond interaction) is as follows, Ecoor>Ecation-π>EH-π≈EH-b>Eπ-π. The coordinate interaction (Ecoor) of His with metallic cations is the strongest interaction with long interaction distance, followed by the cation-π interaction (Ecation-π). In the cation-π interactions, when His is in neutral form (unprotonated), interaction energy is attractive. However, when His is protonated, the interaction energy turns to repulsive. The π-π stacking interactions are the π-plane to π-plane interactions, with much more interaction conformations than other interaction types. In proteins the energies of π-π stacking interactions (Eπ-π) can change in a broad range, because of different interaction orientations. The π-π stacking interactions between neutral His and aromatic amino acids (Phe, Tyr, and Trp) are in the range −3.0 to −4.0 kcal/mol, significantly larger than the van der Waals interactions. However, the π-π stacking energies of protonated histidine (His+) are much larger than the energies of neutral His.
The interaction strength of cation-π interactions in solutions is a controversial research topic [17, 65, 69]. Based on our calculations by using PCM method, the energies of cation-π interactions decrease sharply with the increase of the dielectric constant ε of solvents. In gas phase the cation-π interaction energies of metallic cations are larger than that of organic cations (Lys+ and Arg+). However, in solutions of polar solvents (water and acetonitrile) the cation-π interaction energies of organic cations (protonated amino acids) are lager than that of metallic cations. The PCM is a continuum medium model [50, 51, 52, 53]. The calculated values of PCM may be not very accurate, but the qualitative order is meaningful. In aqueous solution the cation-π interactions between protonated amino acids and aromatic amino acids may be more important than that of metallic cations [17, 69, 70]. However, this does not mean that the cation-π interactions of metallic cations are not important in solutions. In aqueous solution the hydrophilic residues are explored on the surface, and the hydrophobic residues are hidden in the core region of protein structures. In the hydrophobic pockets of proteins the dielectric constants are smaller than that in bulk solution. Therefore, the cation-π interactions are still working in the hydrophobic pockets and in core region of proteins.
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