Mario V. Zlatović*a,
Sunčica Z. Borozanb,
Milan R. Nikolića and
Srđan Đ. Stojanovićc
aFaculty of Chemistry, University of Belgrade, Belgrade, Serbia. E-mail: mario@chem.bg.ac.rs
bDepartment of Chemistry, Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia
cICTM-Department of Chemistry, University of Belgrade, Belgrade, Serbia
First published on 22nd April 2015
In this work, we have analyzed the influence of anion–π interactions on the stability of high resolution protein–porphyrin complex crystal structures. The anion–π interactions are distance and orientation dependent. Results of ab initio calculations of stabilization energies showed that they lie mostly in the range from −2 to −4 kcal mol−1 with some of the anion–π interactions having stabilization energies of up to −16 kcal mol−1. In the anionic group, the numbers of anion–π interactions involving Asp and Glu are similar, while His is more often involved in these interactions than other aromatic residues. Furthermore, our study showed that in the dataset used about 70% of the investigated anion–π interactions are in fact multiple anion–π interactions. Our results suggest that interacting residues are predominantly located in buried and partially buried regions. The secondary structure of the anion–π interaction involving residues shows that most of the interacting residues preferred to be in helix conformations. Significant numbers of aromatic residues involved in anion–π interactions have one or more stabilization centers, providing additional stability to the protein–porphyrin complexes. The conservation patterns indicate that more than half of the residues involved in these interactions are evolutionarily conserved, indicating that the contribution of the anion–π interaction is an important factor for the structural stability of the investigated protein–porphyrin complexes.
While widely studied in supramolecular assemblies, the investigations of anion–π interactions in biological macromolecules and their role therein is still on its beginning. Some studies indicated that such interactions may be of importance in protein structures. A systematic search through the Protein Data Bank (PDB) showed for the first time that anion–π close contacts exist in protein structures between the standard aromatic residues (Trp, Phe, Tyr, His) and anions, such as chloride and phosphate.8 Hinde and co-workers also performed a PDB search focusing on interactions between Phe and negatively charged residues such as Asp and Glu. While edgewise interactions (in which the angle between the anion group and the plane of the ring ranges between 0 and 40°) were found to be very common and significantly attractive (estimated energies range between −8 and −2 kcal mol−1), anion–π interactions involving the ring face were found less frequently and with energies close to zero (weakly attractive or slightly repulsive).14 Also, by a systematic search of protein structures followed by ab initio calculations, Deyà and co-workers showed that anion–π interactions are likely to occur in flavin-dependent enzymes.15 In addition, Moore and co-workers have examined high-resolution structures of proteins and nucleic acids for the presence of “η6”-type anion–π contacts,10 when the anion is directly above the six-membered ring center. Anion–π interactions are now beneficially exploited in fields such as anion sensing,16,17 anion transport through membranes,18,19 or supramolecular assembly,20–22 and they are even considered relevant for anion transport in biological systems.15 Such synthetic channels are of great interest in light of the importance of anion channels in diseases such as cystic fibrosis and other anion channelopathies.23 Recently, Sacchettini and co-workers reported an outstanding study on the development of effective antituberculosis drugs, where anion–π interactions play an important role.24 In spite of the increasing experimental evidences of anion–π interaction, however, study of molecular self-assembly with anion–π interaction as a guiding force is very rare.25,26 In our recently published manuscript, we proposed that an anion–π interactions can contribute significantly to Sm/LSm protein stabilization.27 Sm/LSm proteins are a family of RNA-binding proteins found in virtually every cellular organism.
Porphyrins are heterocyclic macrocycles composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges. In addition, porphyrins are aromatic conjugate acids of ligands that bind metals to form complexes. Some iron-containing porphyrins are called hemes.28,29 Porphyrin-containing proteins are involved in many different processes in living organisms, including oxygen binding, electron transfer, signaling function and catalysis. For example, porphyrin-containing proteins are constituents of photosynthetic reaction centers. A light-harvesting antenna complex is a complex of subunit proteins that may be part of a larger supercomplex of a photosystem, the functional unit in photosynthesis. It is used by plants and photosynthetic bacteria to collect more of the incoming light than could be captured by the photosynthetic reaction center alone using resonance energy transfer.30–35 Understanding porphyrin recognition and its interactions with protein provides insight into how structures are related to porphyrin biological functions.
This manuscript expands on our previous work on the non-canonical interactions of porphyrins in porphyrin-containing proteins36,37 by analyzing the same class of proteins with respect to anion–π interactions. The characteristic features of residues involved in anion–π interactions have been evaluated in terms of the distribution of anion–π interactions, interaction geometries, energetic contribution, solvent accessibility, secondary structure preference, stabilizing centers and conservation score of interacting residues. We have focused our study at the protein–porphyrin interface and hence the anion–π interactions within a protein are not considered. Results from this study stress the importance of anion–π interacting residues in the structural stability and specificity of protein–porphyrin complexes.
Calculations were performed using Jaguar from Schrödinger Suite 2014-3.42 All calculations were performed in vacuum. For ab initio calculations, LMP2 method with triple zeta Dunning's correlation consistent basis set43 and ++ diffuse functions44 was used. The LMP2 method applied to the study of anion–π interactions is considerably faster than the MP2 method, and the interaction energies and equilibrium distances are almost identical for the two methods.45
Geometries of mimetic structures 1–6 were optimized using LMP2/cc-pVTZ(-f)++ level of theory and their single point energies calculated at LMP2/cc-pVTZ++ level. Optimized geometries of mimetic structures 1–6 were placed in space to yield corresponding complex by superimposing matching heavy atoms to their respective coordinates from crystal structures. Then, the energies of complexes produced in that way were calculated.
The interaction energies of the complexes (anion–π pairs) were computed as the difference between the energy of the complex and the sum of the energies of the monomers in their optimized geometries.
Na | %b | Nanion–πc | %anion–πd | |
---|---|---|---|---|
a The number of amino acid in whole database.b Percent of amino acid in whole database.c Number of anion–π interactions in protein–porphyrin complexes.d Percent of anion–π interactions in protein–porphyrin complexes. | ||||
Asp | 740 | 6.2 | 25 | 8.9 |
Glu | 720 | 6.0 | 26 | 9.3 |
His | 402 | 3.4 | 79 | 28.1 |
Phe | 542 | 4.5 | 60 | 21.4 |
Trp | 162 | 1.4 | 31 | 11.0 |
Tyr | 365 | 3.1 | 49 | 17.4 |
RCOO− (porphyrin)–pyrrole (porphyrin) | — | — | 11 | 3.9 |
Total | 2931 | 24.6 | 281 | 100 |
The ratio of Asp to Glu involved in anion–π pairs is very close to the ratio of Asp to Glu residues observed in our entire database (Table 1).
Amongst the aromatic residues, we observed that Phe has the highest occurrence in porphyrin-containing proteins. The contribution of His and Tyr is somewhat smaller than Phe whereas Trp has the lowest occurrence in the dataset studied. The analysis has shown that most carboxylates (RCOO−) of acetyl and propionate groups of porphyrins can be involved in anion–π interactions with surrounding protein aromatic residues. Besides, in analyzing proteins of the present database, we have found interactions between acidic amino acids (Asp and Glu) and π systems of porphyrin (pyrrole ring). No apparent preference for Asp or Glu exists in their interaction with porphyrin ring. The occurrence of His, Phe and Tyr residues in anion–π interactions are 28%, 21% and 17% respectively (Table 1). It was curious to note that Trp, which in the whole database appears with a frequency of 1.4%, has almost the same abundance of interactions as Tyr. The lowest frequency of involvement (11%) in anion–π interactions by Trp residue can be explained by the fact that Trp is the least frequently occurring amino acid in any protein.52 Considering the aromatic residues, His is the most common amino acid involved in such interactions. The unique structure of histidine makes it plays multiple roles in the molecular interactions. While histidine is aromatic and could engage in stacking interactions, it also has the possibility of being protonated and participating in a cation–π interaction.53 Generally the composition of anion–π interaction forming residues is similar to Sm/LSm proteins.27
It is very interesting to note that in the proteins that contain more than one porphyrin, the carboxylate groups of porphyrin can be involved in anion–π interactions with aromatic pyrrole groups of another porphyrin in the protein. We have found 11 (3.9%) of those interactions. An illustrative example of anion–π interactions between two iron–porphyrins from the binding pocket of the cytochrome c from Shewanella oneidensis MR1 (PDB ID: 1m1q) is shown in Fig. 3. There are two anion–π interactions between the porphyrins (HEM802:O2A—HEM803:PyrroleA, HEM802:O2D—HEM803:PyrroleA). Thus, our analysis indicates that the contribution of amino acids toward a particular anion–π interaction is specific in porphyrin-containing proteins. It is likely that these interactions contribute significantly to the overall stability of porphyrin rings.
Fig. 3 Details of the interactions linking the two porphyrins of the cytochrome c from Shewanella oneidensis MR1 (PDB ID: 1m1q). The anion–π interactions are marked with brown dashed lines (HEM802:O2A—HEM803:PyrroleA, HEM802:O2D—HEM803:PyrroleA). Figure was prepared using the program Discovery Studio Visualizer 4.1.40 |
Our database search found that aromatic systems and anions in protein structures are frequently involved in various multiple interactions, including the multiple anion–π interactions. An illustrative examples are shown in Fig. 4. An anion group from propionates (cytochrome c1 from Saccharomyces cerevisiae; PDB ID: 3cx5) can interact with five- and six-membered rings of tryptophan simultaneously (Fig. 4a). The anion–π interactions are marked with brown dashed lines (C:HEM4002:O2A—C:Trp30, C:HEM4002:O2D—C:Trp30). Very interestingly, many protein crystal structures demonstrate that an anion is capable of binding with several aromatic residues. For example, such an interaction motif (Fig. 4b) exists in the crystal structure of a human uroporphyrinogen decarboxylase (PDB ID: 1r3w). An anion group from Asp86 is surrounded by four aromatic pyrroles (A:ASP86:OD2—A:CP3950:PyrroleA, A:ASP86:OD2—A:CP3950:PyrroleB, A:ASP86:OD2—A:CP3950:PyrroleC, A:ASP86:OD2—A:CP3950:PyrroleD).
Fig. 4 Details of multiple anion–π interactions. (a) Several anions clustering around an aromatic group (PDB ID: 3cx5). (b) An anion with multiple aromatics (PDB ID: 1r3w). The anion–π interactions are marked with brown dashed lines. Figure was prepared using the program Discovery Studio Visualizer 4.1.40 |
The analysis shows that around 70% of the total interacting residues in the dataset are involved in the formation of multiple anion–π interactions. This emphasizes previous findings that furcation is an inherent characteristic of macromolecular crystal structures.37 The importance of multiple non-covalent weak interactions, including the anion–π interactions, for governing multicomponent supramolecular assemblies has been already reported.13 Another additional feature is previously noticed additive property of anion–π interactions, showing an effect on the strength of the host–guest system.4,54 Those interactions showed as approximately additive, going from single anion–π to ternary anion–π and even to quaternary anion–π complexes.55
The geometrical details of residues forming anion–π interactions are quantified in terms of the parameters (R, θ) described in the Methodology section. The frequency distribution of the distance and angle parameters of anion–π interacting pairs were analyzed (Fig. 5). Fig. 5a shows that these pairs predominantly occur when the residues are separated by a distance of 4.5 Å or larger. The distribution of distances was found to be bimodal with a prominent maxima at 4.75 and 6.75 Å, corresponding to single and multiple anion–π interactions, respectively. The reason for that is that single interactions have a greater flexibility. The aromatic ring–carboxylate angles were distributed between all angles (0 to 90° range), with a preference for higher angle values (Fig. 5b). The number of pairs increases as θ increases, and more pairs are observed at larger θ values. A distribution of the angles below 20° shows coplanarity, possibly to maximize π–π stacking and packing,10,58 while axial aromatic–anionic pairs are more likely (θ > 60°). There is a no significant statistical difference in the angle distribution between the multiple and the single anion–π interactions. In general, anion–π contacts are realized all over the π system. Similar trends were observed in our earlier study of a Sm/LSm ensemble of proteins from the PDB.27
Fig. 5 Distance (a) and θ angle (b) distribution of anion–π interactions in protein–porphyrin complexes found to form 281 anion–π interactions. |
The geometries that are observed in abundance are not necessarily the ones that have the highest interaction energy between the two moieties in a pair, but the ones that can provide the maximum overall stability to the protein structure by the optimum use of all anion–π interactions. Therefore, we have analyzed the interaction energy of the different anion–π pairs identified in protein–porphyrin complexes. Within a large protein structure numerous interaction modes are possible, and a single binding energy calculation cannot easily isolate which of these are present and their relative importance to overall stabilization. Therefore, it is difficult to parse out the role of the anion–π interaction in their energetics, and the interacting pair residues participating in other noncovalent interactions were not analyzed. In our database it was found that anion–π interactions showed energy less than −16 kcal mol−1, and most of them have energy in the range −2 to −4 kcal mol−1. It has been reported that in thousands of protein PDB structures between Phe and negatively charged residues such as Asp and Glu, anion–π interactions have an energy less than −8 kcal mol−1.14,27
We have calculated the interaction energy for all possible interacting pairs and the results are presented in Fig. 6. The energy of anion–π interaction depends upon various factors such as the size and electronic structure of the anion, nature of the π-ligand, the directionality, and interplay with other noncovalent interactions.4,13 In investigated group, in terms of energetically significance, the energies from His interactions showed to be higher when compared to energies of other groups (Phe, Trp, Tyr and porphyrin) (Fig. 6). It is interesting to note that even in the absence of highly electron-withdrawing groups in the aromatic ring (Phe, Trp) we observe that the strongest interaction energies were associated with edgewise interactions. This pattern arises from the positive electrostatic potential at the ring edge compared to a negative electrostatic potential at the ring face associated with the π electron clouds (Fig. 7). The preference of the anion position can be altered to above the ring if the π-system is electron deficient (His). Nitrogen-containing arenes are electron deficient; consequently, they exhibit the ability to bind anions (through anion–π contacts). The central area of histidine has a higher positive potential than other aromatic rings (Fig. 7) due to the electron withdrawing nitrogen atom. It is notable, however, that in His, Trp and porphyrin rings, there is a substantial area of positive charge concentrated on the nitrogen atoms, which renders the molecules good candidates for establishing anion–π interactions.
Fig. 7 ESPs mapped onto electron density isosurfaces for mimetic structures: 5-methyl-1H-imidazole (His), methylbenzene (Phe), 3-methyl-1H-indole (Trp), 4-methyphenol (Tyr), and 3,4-dimethyl-1H-pyrrole (porphyrin). Typically, a color scale is used, with the most negative potential colored red and the most positive potential colored violet. Electrostatic potential surface energies range is shown below the maps. Figure was prepared using the program Jaguar from Schrödinger Suite 2014-3.42 |
We have observed that most of the anion–π pairs have energies in the range from −2 to −4 kcal mol−1. There are a few residues that have energies less than −16 kcal mol−1, consistent with shorter distance to nitrogen atoms from His, Trp and porphyrin rings (Fig. 6a, c and e). These structures demonstrate high electropositive character (colored violet) on the nitrogen atom area and the establishment of favourable anion–π interactions (Fig. 7). Our ab initio calculations of interacting forces for anion–π structures showed that the strongest attractive interactions (−16 kcal mol−1) emerges between Asp86 and aromatic pyrroles in human uroporphyrinogen decarboxylase; PDB ID 1r3w (Fig. 4b).
The energies of many of the anion–π interactions are quite substantial, but roughly one third of the interactions found showed destabilizing energies (positive values) for dimeric model structures examined in this research (Fig. 6). Although that type of interactions, observed under isolated conditions, as in this research, can be considered to weaken the stability of protein structure, this has to be taken with some dose of reserve. Namely, we mentioned earlier that the database search showed that aromatic systems and anions in protein structures are frequently involved in various multiple long range non-covalent interactions (Fig. 8), as well as the fact that non-covalent interactions are additive in their nature. The combination of the anion–π interaction with other type(s) of non-covalent bonds can work in a synergistic manner and show to be desirable for achieving the stability of systems.13 However, the precise nature and quantitative interaction energies of those multiple interactions working in synergy and the factors affecting their interaction energies still needs further investigations.
Fig. 8 Details of anion–π interactions with destabilizing energies of the nonaheme cytochrome c from Desulfovibrio desulfuricans Essex (PDB ID: 1duw). (a) Energy of anion–π interaction A:HEM301:O2D—A:His200 (2.11 kcal mol−1) is compensated from coordinated bond (His200–Fe2+). (b) Energy of anion–π interaction A:Glu41:OE2—A:HEM296(B) (2.18 kcal mol−1) is compensated from other non-covalent interactions. Hydrophobic interactions are missed for clarity images. Figure was prepared using the program Discovery Studio Visualizer 4.1.40 |
To understand the interactions that confer secondary structural conformational stability in proteins it is important to know the conformational preferences of amino acids. In order to obtain the preference and pattern of each anion–π interaction forming residue in protein–porphyrin complexes, we conducted a systematic analysis based on their location in different secondary structures. We have calculated the occurrence of anion–π interaction forming residues in different secondary structures in our dataset which classify amino acids into helix, strand, and turn, and the results are presented in Table 2.
Amino acid | Helixa (%) | Helixb (%) | Stranda (%) | Strandb (%) | Turna (%) | Turnb (%) |
---|---|---|---|---|---|---|
a Percent of secondary structure residues in whole database.b Percent of secondary structure residues involved in anion–π interactions. | ||||||
Asp | 40.0 | 50.0 | 6.8 | 16.7 | 53.2 | 33.3 |
Glu | 61.9 | 56.0 | 5.3 | 12.0 | 32.8 | 32.0 |
His | 47.5 | 59.0 | 5.0 | 4.9 | 47.5 | 36.1 |
Phe | 53.3 | 26.8 | 9.9 | 25.0 | 36.8 | 48.2 |
Trp | 53.9 | 45.5 | 14.5 | 22.7 | 31.6 | 31.8 |
Tyr | 51.5 | 59.5 | 17.1 | 16.2 | 31.4 | 24.3 |
We have analyzed amino acid secondary structure preferences for the whole dataset of 65 protein chains. Among all amino acids, 45.4% belong to helices, 11.9% to sheets, and 42.7% to turns. Further, we have analyzed the percentage occurrence of the anion–π interactions in a particular secondary structure, irrespective of the amino acid. It was found that most of the anion–π interactions between the residues prefer the secondary structure of helical segments, followed by turn and strand conformations. This is probably due to the fact that helices are more represented than other types of secondary structures. Anionic residues such as Asp and Glu preferred to be in helix. In the aromatic group, we found that His, Trp and Tyr were predominantly in helix as well, while Phe was dominantly in turn. When we compare the occurrence of the amino acids in a particular secondary structure in whole dataset and anion–π interaction forming set, we can notice that there is similar trend. It was interesting to observe that a significant percentage of Asp, Glu and Phe residues favoured strand conformation while form anion–π interactions. Our data are consistent with the results observed with anion–π pairs in a nonredundant, high-resolution subset of the Protein Data Bank14 observed that the anion–π interactions is well represented among inter-helical interactions. Hence, the preference of an amino acid to form anion–π interactions in particular secondary structure is not the same as the preference of the amino acid for a particular secondary structure.60
We have computed the stabilization centers for all anion–π interaction forming residues in protein–porphyrin complexes. Table 3 shows the percentage contribution of the individual amino acid residue which is part of the stabilizing center involved in anion–π interactions.
Amino acid | Nanion–πa | SCb | SC%c |
---|---|---|---|
a Number of anion–π interactions in protein–porphyrin complexes.b Number of SC residues involved in anion–π interactions.c Percent of SC residues involved in anion–π interactions. | |||
Anionic | |||
Asp | 25 | 6 | 24.0 |
Glu | 26 | 6 | 23.1 |
Total | 51 | 12 | 23.5 |
π residues | |||
His | 79 | 26 | 32.9 |
Phe | 60 | 11 | 18.3 |
Trp | 31 | 14 | 45.2 |
Tyr | 49 | 19 | 38.8 |
Total | 219 | 70 | 32.0 |
Considering the whole dataset, 82 (30.4%) stabilizing residues are involved in building anion–π interactions. Aromatic residues were found to have more stabilization centers than anionic residues. It was found that 23.5% of anionic residues and 32.0% of π residues were found to have one or more stabilization centers. Among the stabilization centers involving π residues Trp was included in more stabilization centers than other residues (45.2%), while Phe showed the least contribution (18.3%). This trend was somewhat different than the earlier report on Sm/LSm proteins.27 It was interesting to note that all the six residues found in anion–π interactions are important in locating one or more stabilization centers. These observations strongly reveal that these residues may contribute significantly to the structural stability of these proteins in addition to participating in anion–π interactions.
Hence we could conclude that, the anion–π interactions are an important factor for the structural stability of the protein–porphyrin complexes studied in this work, and this study provides a contribution for the development of appropriate theory to accommodate these molecular interactions and their application in molecular design.
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