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“η6”-Type anion–π in biomolecular recognition
FEBS Letters 586 (2012) 4180–4185
journal homepage: www.FEBSLetters.org
‘‘g6’’-Type anion–p in biomolecular recognition Suvobrata Chakravarty a,⇑,1, Zi-Zhang Sheng a,1, Bradley Iverson a, Brian Moore b a b
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA Office of Information Technology, South Dakota State University, Brookings, SD 57007, USA
a r t i c l e
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Article history: Received 30 July 2012 Revised 24 September 2012 Accepted 1 October 2012 Available online 23 October 2012 Edited by Paul Bertone Keywords: Non-covalent interaction Anion–p Cation–p Evolutionary conservation Nucleic acid tetraloop
a b s t r a c t Theoretical and compelling experimental evidence indicates that the interaction between an anion and an aromatic p system when the anion is directly above the ring face (‘‘g6’’-type anion–p), can be attractive. This may play an important role in the formation and recognition of biomolecular structures. We examined high-resolution structures of proteins and nucleic acids for the presence of ‘‘g6’’-type anion–p. Though less frequent than its counterpart cation–p, ‘‘g6’’-type anion–p is observed unambiguously, occurring in protein/nucleic acid loops and often involving conserved/ coevolving sites in proteins, suggesting it plays an important role in macromolecular folding and function. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The assembly of biological molecules is guided by weak noncovalent interactions such as hydrogen bonding, hydrophobic interactions, ion pairs and p–p stacking [1]. However, a number of other weak interactions have been reported [2]. Among them, the interaction between a cation and p systems, known as the cation–p interaction, has overshadowed the others due to its widespread occurrence in proteins and nucleic acids [3]. In a typical cation–p pair observed in proteins, positively charged side-chain atoms, Lys-NZ and Arg-CZ, interact with the p electron cloud on the face of the side-chain aromatic ring (Phe, Tyr, and Trp). Though aromatic rings (Phe, Tyr, and Trp) are non-polar (no net permanent dipole moment), they have quadrupole moments because of the two opposing dipoles originating from each face [4]. Due to these quadrupole moments, cations interact with the aromatic face. While the aromatic face interacts with cations, the ring edges of the quadrupole have now been observed to interact with Asp and Glu carboxylates called the anion–p interaction [5]. In general, anions positioned facing the aromatic ring are believed to be unfavorable [5]. However, systematic studies involving the crystal structures of salts of organic molecules, such as pentafluorophenyl ⇑ Corresponding author. Address: Department of Chemistry and Biochemistry, Box-2202, SAV367, South Dakota State University, Brookings, SD 57007, USA. Fax: +1 605 688 6364. E-mail address: [email protected] (S. Chakravarty). 1 These two authors contributed equally to the work.
rings, have captured anions facing the ring [6]. These studies suggest that changes in electronic behavior (electron withdrawing ring substitution) can accommodate anions on the ring face. These findings have inspired the idea of designing synthetic anion-specific transporters to treat chloride channelopathies, or designing nitrate sensors for monitoring cellular signaling events [7]. To guide design of synthetic molecules for such applications requires appropriate theoretical studies [8]. To better understand such an interaction, we surveyed the existence of anions facing aromatic rings in biological macromolecules for their abundance of anions (Asp, Glu, Phosphates). In the structures of pentafluorophenyl salts [6], the geometry of the observed anion facing the ring plane has been described as ‘‘g1’’, ‘‘g2’’, ‘‘g3’’ or ‘‘g6’’ based on the anion’s proximity to a number of the six-membered ring’s carbon atoms, where ‘‘g6’’ has the anion above the ring center. In this analysis, we loosely define the ‘‘g6’’-type of anion–p as occurring whenever an anion is nearly perpendicular to the ring plain within a specified distance d 6 4.5 Å from the ring center and subtending an angle h 6 15° with the ring normal (Fig. 1a). This distance is at the upper end of those observed in organic salt crystal structures [6]. We observe the presence of such interacting pairs in high-resolution structures of proteins, RNA and RNA–protein complexes, suggesting nature’s utilization of yet another type of non-covalent interaction. We systematically analyze occurrences of ‘‘g6’’-type anion–p in biological macromolecules and compare these interactions with cation–p to obtain a better understanding of nature’s design principles.
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.10.017
S. Chakravarty et al. / FEBS Letters 586 (2012) 4180–4185
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Fig. 1. ‘‘g6’’-Type anion–p in biomolecules: (a) cartoons representing the detection criterion (left) and the anions (carboxylate, middle; phosphate, right) discussed in this study. For all figures, the aromatic residue carbon atoms are colored purple while the carboxylate carbon atom(s) of the anion are gray (b) Transketolase (pdbid 2r8o) anion–p (left) and the distribution of the inter-plane angle between the carboxylate and the aromatic ring (right) (c) hydrogen-bonded (left) and non-hydrogen-bonded anion–p (right).
2. Materials and methods 2.1. Construction of the dataset A non-redundant set consisting of 1560 protein single chains was chosen from Cull-PDB [9]. The sequence identity between any two chains in this set is 625% and their structures have been determined with a resolution 61.6 Å and an R factor of 0.25. PDB files that have coordinates missing for Asp, Glu, Phe, Trp, Tyr, Lys, Arg, Asn or Gln residues were eliminated in the construction of the final set, which consisted of 1059 single chains. For evolutionary conservation analysis, another set of PDB chains was constructed such that chain sequences belonged to the PFAM [10] protein domain family. For this set, only 246 PFAM [10] families provided at least one structure with resolution 61.5 Å and had more than 300 sequences in the PFAM alignment (after removal of redundant sequences at 85% sequence identity by CD-Hit [11]). For analysis of RNA, DNA and RNA/DNA protein complexes, the sequence information in the coordinate files of the entire PDB dataset was scanned for residues A, T, G, C and U. Any coordinate file with residues A, T, G, C and U was set aside and sequence
information was manually checked to make sure the structure contained nucleic acids. The resolution of such structures were not as high as those of cull-PDB, and our analysis was performed on nucleic acid structures with resolutions 62.5 Å. 2.2. Analysis procedure The aromatic ring gravity centers of Phe, Tyr and Trp residue side-chains were determined. The unit vectors normal to these ring planes were determined (see Supplemental Information for details). For protein anions, we assumed that Asp, Glu carboxylate ions are ionized, and the distance, ro, between a carboxylate oxygen atom and the aromatic ring centers was computed. A distance cutoff |ro| 6 4.5 Å was used to select anion–p pairs. A cutoff for the angle h 6 15° between ro and the normal to the ring was imposed to eliminate anions interacting with the ring edges. For the nucleic acid phosphates, each of the two non-ester oxygen atoms was treated as anion, and for the nucleic acid base ring gravity center only the ring atoms were considered, disregarding the amino and the carbonyl oxygen atoms connected to the nucleobase ring.
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3. Results and discussion 3.1. ‘‘g6’’-Type anion–p geometry Using the distance-angle criteria (see Section 2), we detected 111 ‘‘g6’’-type anion–p interacting pairs in the dataset. With the same criterion, we observed 301 cation–p pairs in the dataset, suggesting that ‘‘g6’’-type anion–p interactions are rarer but unambiguously present in protein structures. For the ‘‘g6’’-type anion– p interactions, the observed distances between pairs were as low as 2.7 Å (Fig. 1b, left) and are comparable to those of the crystal structures of organic salts [6]. B-factors of the ‘‘g6’’-type anion–p interacting atoms were well below 20 (Fig. S1a), suggesting minimal positional ambiguity for these atoms. A distribution of the angle between the carboxylate ion and the ring plane shows that co-planarity is preferred (Fig. 1b, right), possibly to maximize p–p stacking and packing [12]. Even if ‘‘g6’’-type anion–p interactions are considered unfavorable under isolated conditions [5], one can argue that the anion could be held above the ring plane by energetic compensation from other interactions involving the rest of the polypeptide chain. This is difficult to address, due to the need to break down the complete energetic components of the entire folded structure. However, we analyzed the possibility of local polar interactions such as hydrogen bonding or salt-bridges directly involving the anion that register it above the aromatic plane (Fig. 1c). Approximately 70% of the ‘‘g6’’-type anion–p carboxylates are either hydrogen-bonded or are involved in ion-pairs, while the remaining 30% are not, suggesting that this interaction
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pair may not always require compensation. Approximately 60% of the cation–p pairs in the set are hydrogen-bonded or involved in ion-pairs, i.e., even attractively interacting pairs are involved in a network of interactions and the ‘‘g6’’-type anion–p observed here may be exhibiting similar behavior. The depth distribution [13] of the atoms involved in ‘‘g6’’-type anion–p and cation–p pairs (Fig. S1b) suggests that the majority of these are just below the surface, i.e., they are partially buried and the energetics of desolvation discussed earlier [4] for cation–p interactions may similarly be applicable to the ‘‘g6’’-type anion–p interaction. Packing is often a good descriptor of the environment around an atom, as packing is influenced by solvent accessibility, secondary structure and other factors [14]. Using the occluded surface and fraction ray length [15], we computed packing in the neighborhood of this interaction. A smaller fraction ray length indicates better packing, while a longer ray length indicates the opposite. The distribution of the fraction ray length of all Asp + Glu pairs in the dataset is a normal distribution (mean ± S.D. of 0.295 ± 0.049) while that of Asp + Glu pairs involved in ‘‘g6’’-type anion–p is very similar to the that of all Asp + Glu (Fig. 2a, left). This indicates that packing Asp + Glu carboxylates on the ring face does not compromise packing. The same can also be concluded from the packing of aromatic residues involved in ‘‘g6’’-type anion–p (data not shown). As the side-chains of aromatic residues and Asp/Glu tend to pack better than amino acids with aliphatic carbons [14], the formation of ‘‘g6’’-type anion–p interactions will result in well-packed side-chains. In other words, formation of ‘‘g6’’-type anion–p interactions may favorably contribute to packing.
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Fig. 2. Features of ‘‘g6’’-type anion–p in biomolecules: (a) distribution of fraction ray length, a measure of the residue packing density, of carboxylate ions involved in ‘‘g6’’type anion–p (gray bars). This distribution is compared with that of any carboxylate ion (black line) in the protein structure. (b) Inter-residue sequence separation between ‘‘g6’’-type anion–p interacting pairs (left, gray bar) and cation–p (right, black bar). (c) ‘‘g6’’-type anion–p in nucleic acids where the base of the ith nucleotide (purple) pairs with the oxygen atoms of an (i + 2)th phosphate (gray) anion.
S. Chakravarty et al. / FEBS Letters 586 (2012) 4180–4185
Distribution of the inter-residue separation between the anion and aromatic residues in the primary structure show that ‘‘g6’’-type anion–p interactions occur preferentially between positions that are very close in the sequence, especially between adjacent residues (i, i + 1) or (i, i 1) (Fig. 2b, left). The ‘‘g6’’-type adjacent anion–p pairs are usually in regions with loops and turns (see example Fig. 1c right) and may favorably influence the local folding of polypeptide chains [16] (see text of Fig. S1). 3.2. ‘‘g6’’-Type anion–p in nucleic acids We also examined the existence of ‘‘g6’’-type anion–p in nucleic acids. Similar to proteins, we observed such interactions in nucleic acid loop/hairpin structures (Fig. 2c). In nucleic acid backbones, such as tetraloop hairpins, the base of the ith nucleotide pairs with the oxygen atoms of the (i + 2)th phosphate anion, forming ‘‘g6’’-type anion–p. The distances of the ‘‘g6’’-type anion–p interactions in the nucleic backbone cases are often below 3 Å. While the rest of the phosphate anionic oxygen atoms in the nucleic acid backbone face outward, the ‘‘g6’’-type phosphate anions (poorly solvated) face inward (Fig. 2c). The solvent accessible surface area of inward (OP2 atom) and outward facing phosphate oxygen atoms are 1.5 Å2 and >20 Å2, respectively, suggesting that the nucleic acid base may compensate for the energetics of the desolvation of the anion. In many RNA hairpins, even in structures with low resolutions (P3 Å) (Fig. S2 and associated text), we observed this interaction between the ith base and the (i + 2)th phosphate consistent with the interactions observed in high resolution structures of Escherichia coli 4.5S RNA tetraloops or the hairpin ribozyme stem C loop (Fig. 2c). This suggests that ‘‘g6’’-type anion–p interactions may be a regular feature of nucleic acid loops. In addition, in a few low resolution RNA structures, we also observed ‘‘g6’’-type anion–p between the ith base and the (i 1)th phosphate oxygen anions (Fig. S2), as well as between distant positions in the primary sequence.
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3.3. ‘‘g6’’-Type anion–p and residue conservation We next examined the evolutionary conservation of positions of interacting pairs in proteins using the relative entropy measure (Fig. S3). A similar trend in the relative entropy is observed for both ‘‘g6’’-type anion–p and cation–p. The relative entropies of the anions and the aromatic residues involved in ‘‘g6’’-type anion–p and the respective pairs involved in cation–p seem to be in the intermediate conservation range (Fig. S3) and may suggest that many of these types of non-covalent interactions can be replaced without compromising the stability of the fold. It is important to note that there is a strong co-evolving trend, i.e., a tendency to retain the partner residue when one of the pairing residues is retained in a protein family (Fig. 3a). This trend is similar for both ‘‘g6’’-type anion–p and cation–p. For example, in the Xylanase structures (pfam Glyco_hydro_10 family), the frequency of the catalytic nucleophile Glu, Asp (Glu241 in pdbid 2q8x, Glu236 in 1e0w) in the alignment column is p(E,D) = 0.591. The frequency Glu, Asp (conditional probability) in a subset of sequences of this family that all have either Trp or Tyr or Phe at the alignment position corresponding to the Trp291 in 2q8x or Trp266 in 1e0w, has the probability p(E,D/ WYF) = 0.996 (Fig. 3a and b). The plot of p(E,D) vs p(E,D/WYF) shows that nearly all of the (x, y) points are above the y = x line (Fig. 3a), and the same is true for the plot of p(W,Y,F) vs p(W,Y,F/ ED), suggesting that natural selection favors conservation of such interacting pairs. In the Xylanase example above, structures of a number of enzymes of the family show that in all cases the catalytic nucleophilic carboxylate is held above the aromatic plane, suggesting that a specific orientation (anti to the endocyclic O5– C1 bond [17]) may be necessary for the hydolysis of the substrate glycosidic linkage. Similarly, Lactococcus lactis branched-chain keto acid decarboxylase (KdcA) (pfam TPP_enzyme_N family) catalytic loop residue Asp26 [18] is involved in an ‘‘g6’’-type anion–p interaction with the adjacent Tyr27 with p(D,E) = 0.25 and p(D,E/ WYF) = 0.892 at position Asp26 (2vbf) (Fig. S3 and the associated
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Fig. 3. Conservation of ‘‘g6’’-type anion–p in biomolecules: (a) plot of p(D,E) vs p(D,E/WYF) (see text) (left) and p(R,K) vs p(R,K/WYF). (b) Sequence logo of a segment of the pfam Glyco_hydro_10 family highlighting the positions involved in ‘‘g6’’-type anion–p (left) and the structure of Xylanase (2q8xB) (right) showing the ‘‘g6’’-type anion–p residues. The secondary structure of this region is shown in blue in Xylanase and is represented below the sequence logo (left) (also see Fig. S3 and associated text).
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text). Some of the examples of co-evolving sites observed here include enzyme catalytic sites. This may suggest that the supporting role of the ‘‘g6’’-type anion–p interaction is in orienting catalytic residues, and could be useful in the design of artificial enzymes that require grafting catalytic residues onto a stable protein scaffold [19]. 3.4. ‘‘g6’’-Type anion–p at interfaces Though no extensive analysis of interfaces is carried out here, we discuss a few interfaces, specifically those involving RNA-protein interactions corresponding to crucial biological processes (Fig. 4). The RRM (RNA recognition motif) of the U1A protein binds to U1 snRNA hairpin II. The structure of the complex reveals that RRM Asp92 stacks in parallel on the Cyt12 residue of the hairpin loop, [20] forming an ‘‘g6’’-type anion–p. The same feature is observed in the structure of the RRM of U2B’’ bound to hairpin IV of U2 snRNP (Fig. 4). U2B’’ Asp92 stacks on Guanine12 of the hairpin loop of U2 and this arrangement seems to be a conserved feature of spliceosomal RNA hairpins involving a continuous stack of four planes of Phe56, A11, C/G12 and Asp92-carboxylates, in which the two amino acid side chains appear to hold the interior stacked bases like clamps at either end [21]. Next we discuss the interaction between tRNA and two distinctly different b-barrel domains. These barrels are the elongation factor isomerase (EI) type of b-barrel in EF-Tu (elongation factor Tu) and the L25-like barrel in amino acyl-tRNA synthetase (aaRS) that binds tRNA. EF-Tu, in constrast to
aaRS, forms complexes with all aa-tRNAs. Hence, EF-Tu recognizes common features of tRNA including the 50 CCA-OH acceptor arm [22]. The terminal 50 -A of the Phe-tRNAPhe acceptor arm stacks against Glu271 of the center b-barrel domain of Thermus acquaticus EF-Tu (Fig. 4). This Glu residue is conserved in the sequences of Homo sapiens, Saccharomyces cerevisiae, E. coli, Bacillus subtilis, Sulfolobus solfataricus, Haloarcula marismortui, and T. aquaticus EF-Tu [23]. This interaction is also observed in the structure of E. coli Cys-tRNACys complexed to EF-Tu [23]. The b-barrel domain of Gln-RS specifically recognizes the anticodon loop of Gln-tRNAGln, enabling Gln-RS to discriminate between cognate and non-cognate tRNAs [24]. The three Gln-tRNA anticodon bases (CUG) are aligned in different directions to fit into base-specific pockets [24]. Glu519 stacks onto U35 in the U35 base-specific pocket. The U35 pocket is thought to be the tightest among the three pockets [24], and packing of the carboxylate ion onto the U35 face may be equally important to the interpretation of the genetic code. A BLAST search (20 iterations, e value cutoff 60.001 and alignment coverage P60%) with the sequence of this domain shows that more that 60% of the retrieved sequences have Glu/Asp residues corresponding to the position of Glu519 in Gln-RS, suggesting strong preference for the anion in the U35 pocket. Other examples of interfacial ‘‘g6’’-type anion–p are provided in the Supplemental Information (Fig. S4). Aromatic residues have long been known for their functional versatility (Fig. S6). With the examples described above, it is clear that the number of versatile functions for aromatic residues in
Fig. 4. ‘‘g6’’-Type anion–p in interfaces: RRM (split-pea green, 1a9n) of U2B00 (spliceosomal machinary) in complex with U2 snRNA (green, top left), EF_TU b-barrel domain (split-pea green, 1b23) in complex with the acceptor arm of Phe-tRNAPhe (green, top right), Glutamyl tRNA synthetase b-barrel domain (split-pea green, 1gtr) in complex with the anticodon arm of Gln-tRNAGln (green, bottom left), Syntenin tandem PDZ (green, 1obx) in complex with IL5Ra (orange, bottom right) (also see Fig. S4 and associated text).
S. Chakravarty et al. / FEBS Letters 586 (2012) 4180–4185
macromolecules is increasing. However, in the absence of point mutation studies, the energetics of the ‘‘g6’’-type anion–p interaction remain to be studied in the future, in a manner similar to experiments performed with cation–p interactions in globular proteins [25]. It is encouraging to note that in a number of examples the protein sequence positions involved in ‘‘g6’’-type anion–p are not only conserved, but the interacting pairs also show co-evolving trends suggesting a possible energetic or functional coupling between these positions [26]. This co-evolution is similar to that of cation–p interactions, the energetics of which are detailed by isostearic point mutations in globular proteins that are suggested, on average, to have stabilizing effects [25]. Similarly, one may expect to find that ‘‘g6’’-type anion–p interactions have stabilizing effects on average, although these interactions may be very weak. Lastly, it is interesting to note that even in the absence of highly electron-withdrawing groups in the aromatic ring we observe ‘‘g6’’type anion–p in macromolecules, even contributing to crucial biomolecular recognition. This suggests that we still lack a comprehensive picture of the nature of weak molecular interactions, and this study provides an impetus for the development of appropriate chemistry theory to accommodate these weak molecular interactions and their application in molecular design.
[10]
Acknowledgements
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The study was supported by the SDSU Agricultural Experimentation grant 3AH386 and Biological Control and Analysis by Applied Photonics (BCAAP), as well as the South Dakota 2010 center grant 3SG163 to S.C. The authors thank Adam Hoppe for carefully reading the manuscript and providing helpful suggestions. The authors thank Jay Shore for providing helpful suggestions.
[17]
[6]
[7]
[8]
[9]
[11]
[12] [13] [14]
[15]
[18]
[19]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012.10. 017. References [1] Kim, K.S., Tarakeshwar, P. and Lee, J.Y. (2000) Molecular clusters of pi-systems: theoretical studies of structures spectra, and origin of interaction energies. Chem. Rev. 100, 4145–4186. [2] Jain, A., Ramanathan, V. and Sankararamakrishnan, R. (2009) Lone pair pi interactions between water oxygens and aromatic residues: quantum chemical studies based on high-resolution protein structures and model compounds. Protein Sci. (Protein Society) 18, 595–605. [3] Dougherty, D.A. (1996) Cation–pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271, 163–168. [4] Gallivan, J.P. and Dougherty, D.A. (2000) A computational study of cationp interactions vs salt bridges in aqueous media: implications for protein engineering. J. Am. Chem. Soc. 122, 870–874. [5] Philip, V., Harris, J., Adams, R., Nguyen, D., Spiers, J., Baudry, J., Howell, E.E. and Hinde, R.J. (2011) A survey of aspartate-phenylalanine and glutamate-
[20]
[21]
[22]
[23]
[24] [25]
[26]
4185
phenylalanine interactions in the protein data bank: searching for anion–pi pairs. Biochemistry 50, 2939–2950. Albrecht, M., Wessel, C., de Groot, M., Rissanen, K. and Luchow, A. (2008) Structural versatility of anion–pi interactions in halide salts with pentafluorophenyl substituted cations. J. Am. Chem. Soc. 130, 4600–4601. Dawson, R.E., Hennig, A., Weimann, D.P., Emery, D., Ravikumar, V., Montenegro, J., Takeuchi, T., Gabutti, S., Mayor, M., Mareda, J., Schalley, C.A. and Matile, S. (2010) Experimental evidence for the functional relevance of anion–pi interactions. Nat. Chem. 2, 533–538. Escudero, D., Frontera, A., Quinonero, D. and Deya, P.M. (2009) Interplay between anion–pi and hydrogen bonding interactions. J. Comput. Chem. 30, 75–82. Wang, G. and Dunbrack Jr., R.L. (2003) PISCES: a protein sequence culling server. Bioinformatics 19, 1589–1591. Punta, M., Coggill, P.C., Eberhardt, R.Y., Mistry, J., Tate, J., Boursnell, C., Pang, N., Forslund, K., Ceric, G., Clements, J., Heger, A., Holm, L., Sonnhammer, E.L., Eddy, S.R., Bateman, A. and Finn, R.D. (2012) The Pfam protein families database. Nucleic Acids Res. 40, D290–301. Li, W. and Godzik, A. (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659. Marsili, S., Chelli, R., Schettino, V. and Procacci, P. (2008) Thermodynamics of stacking interactions in proteins. Phys. Chem. Chem. Phys. 10, 2673–2685. Chakravarty, S. and Varadarajan, R. (1999) Residue depth: a novel parameter for the analysis of protein structure and stability. Structure 7, 723–732. Fleming, P.J. and Richards, F.M. (2000) Protein packing: dependence on protein size, secondary structure and amino acid composition. J. Mol. Biol. 299, 487– 498. Pattabiraman, N., Ward, K.B. and Fleming, P.J. (1995) Occluded molecular surface: analysis of protein packing. J. Mol. Recognit. 8, 334–344. Baldwin, R.L. and Rose, G.D. (1999) Is protein folding hierarchic? II. Folding intermediates and transition states (vol. 24, p. 77, 1999). Trends Biochem. Sci. 24, 185–186. Heightman, T.D. and Vasella, A.T. (1999) Recent insights into inhibition, structure, and mechanism of configuration-retaining glycosidases. Angew. Chem., Int. Ed. 38, 750–770. Berthold, C.L., Gocke, D., Wood, D., Leeper, F.J., Pohl, M. and Schneider, G. (2007) Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction. Acta Crystallogr., Sect. D: Biol. Crystallogr. 63, 1217–1224. Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Rothlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L. and Baker, D. (2008) De novo computational design of retroaldol enzymes. Science 319, 1387–1391. Oubridge, C., Ito, N., Evans, P.R., Teo, C.H. and Nagai, K. (1994) Crystal-structure at 1.92 Angstrom resolution of the RNA-binding domain of the U1a spliceosomal protein complexed with an RNA hairpin. Nature 372, 432–438. Price, S.R., Evens, P.R. and Nagai, K. (1998) Crystal structure of the spliceosomal U2B00 –U2A0 protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650. Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F.C. and Nyborg, J. (1995) Crystal-structure of the ternary complex of PheTrna(Phe), Ef-Tu, and a Gtp Analog. Science 270, 1464–1472. Nissen, P., Thirup, S., Kjeldgaard, M. and Nyborg, J. (1999) The crystal structure of Cys-tRNA(Cys)-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA. Struct. Fold. Des. 7, 143–156. Rould, M.A., Perona, J.J. and Steitz, T.A. (1991) Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature 352, 213–218. Prajapati, R.S., Sirajuddin, M., Durani, V., Sreeramulu, S. and Varadarajan, R. (2006) Contribution of cation–pi interactions to protein stability. Biochemistry 45, 15000–15010. Lockless, S.W. and Ranganathan, R. (1999) Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299.
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‘‘g6’’-Type anion–p in biomolecular recognition Suvobrata Chakravarty a,⇑,1, Zi-Zhang Sheng a,1, Bradley Iverson a, Brian Moore b a b
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA Office of Information Technology, South Dakota State University, Brookings, SD 57007, USA
a r t i c l e
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Article history: Received 30 July 2012 Revised 24 September 2012 Accepted 1 October 2012 Available online 23 October 2012 Edited by Paul Bertone Keywords: Non-covalent interaction Anion–p Cation–p Evolutionary conservation Nucleic acid tetraloop
a b s t r a c t Theoretical and compelling experimental evidence indicates that the interaction between an anion and an aromatic p system when the anion is directly above the ring face (‘‘g6’’-type anion–p), can be attractive. This may play an important role in the formation and recognition of biomolecular structures. We examined high-resolution structures of proteins and nucleic acids for the presence of ‘‘g6’’-type anion–p. Though less frequent than its counterpart cation–p, ‘‘g6’’-type anion–p is observed unambiguously, occurring in protein/nucleic acid loops and often involving conserved/ coevolving sites in proteins, suggesting it plays an important role in macromolecular folding and function. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The assembly of biological molecules is guided by weak noncovalent interactions such as hydrogen bonding, hydrophobic interactions, ion pairs and p–p stacking [1]. However, a number of other weak interactions have been reported [2]. Among them, the interaction between a cation and p systems, known as the cation–p interaction, has overshadowed the others due to its widespread occurrence in proteins and nucleic acids [3]. In a typical cation–p pair observed in proteins, positively charged side-chain atoms, Lys-NZ and Arg-CZ, interact with the p electron cloud on the face of the side-chain aromatic ring (Phe, Tyr, and Trp). Though aromatic rings (Phe, Tyr, and Trp) are non-polar (no net permanent dipole moment), they have quadrupole moments because of the two opposing dipoles originating from each face [4]. Due to these quadrupole moments, cations interact with the aromatic face. While the aromatic face interacts with cations, the ring edges of the quadrupole have now been observed to interact with Asp and Glu carboxylates called the anion–p interaction [5]. In general, anions positioned facing the aromatic ring are believed to be unfavorable [5]. However, systematic studies involving the crystal structures of salts of organic molecules, such as pentafluorophenyl ⇑ Corresponding author. Address: Department of Chemistry and Biochemistry, Box-2202, SAV367, South Dakota State University, Brookings, SD 57007, USA. Fax: +1 605 688 6364. E-mail address: [email protected] (S. Chakravarty). 1 These two authors contributed equally to the work.
rings, have captured anions facing the ring [6]. These studies suggest that changes in electronic behavior (electron withdrawing ring substitution) can accommodate anions on the ring face. These findings have inspired the idea of designing synthetic anion-specific transporters to treat chloride channelopathies, or designing nitrate sensors for monitoring cellular signaling events [7]. To guide design of synthetic molecules for such applications requires appropriate theoretical studies [8]. To better understand such an interaction, we surveyed the existence of anions facing aromatic rings in biological macromolecules for their abundance of anions (Asp, Glu, Phosphates). In the structures of pentafluorophenyl salts [6], the geometry of the observed anion facing the ring plane has been described as ‘‘g1’’, ‘‘g2’’, ‘‘g3’’ or ‘‘g6’’ based on the anion’s proximity to a number of the six-membered ring’s carbon atoms, where ‘‘g6’’ has the anion above the ring center. In this analysis, we loosely define the ‘‘g6’’-type of anion–p as occurring whenever an anion is nearly perpendicular to the ring plain within a specified distance d 6 4.5 Å from the ring center and subtending an angle h 6 15° with the ring normal (Fig. 1a). This distance is at the upper end of those observed in organic salt crystal structures [6]. We observe the presence of such interacting pairs in high-resolution structures of proteins, RNA and RNA–protein complexes, suggesting nature’s utilization of yet another type of non-covalent interaction. We systematically analyze occurrences of ‘‘g6’’-type anion–p in biological macromolecules and compare these interactions with cation–p to obtain a better understanding of nature’s design principles.
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.10.017
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Fig. 1. ‘‘g6’’-Type anion–p in biomolecules: (a) cartoons representing the detection criterion (left) and the anions (carboxylate, middle; phosphate, right) discussed in this study. For all figures, the aromatic residue carbon atoms are colored purple while the carboxylate carbon atom(s) of the anion are gray (b) Transketolase (pdbid 2r8o) anion–p (left) and the distribution of the inter-plane angle between the carboxylate and the aromatic ring (right) (c) hydrogen-bonded (left) and non-hydrogen-bonded anion–p (right).
2. Materials and methods 2.1. Construction of the dataset A non-redundant set consisting of 1560 protein single chains was chosen from Cull-PDB [9]. The sequence identity between any two chains in this set is 625% and their structures have been determined with a resolution 61.6 Å and an R factor of 0.25. PDB files that have coordinates missing for Asp, Glu, Phe, Trp, Tyr, Lys, Arg, Asn or Gln residues were eliminated in the construction of the final set, which consisted of 1059 single chains. For evolutionary conservation analysis, another set of PDB chains was constructed such that chain sequences belonged to the PFAM [10] protein domain family. For this set, only 246 PFAM [10] families provided at least one structure with resolution 61.5 Å and had more than 300 sequences in the PFAM alignment (after removal of redundant sequences at 85% sequence identity by CD-Hit [11]). For analysis of RNA, DNA and RNA/DNA protein complexes, the sequence information in the coordinate files of the entire PDB dataset was scanned for residues A, T, G, C and U. Any coordinate file with residues A, T, G, C and U was set aside and sequence
information was manually checked to make sure the structure contained nucleic acids. The resolution of such structures were not as high as those of cull-PDB, and our analysis was performed on nucleic acid structures with resolutions 62.5 Å. 2.2. Analysis procedure The aromatic ring gravity centers of Phe, Tyr and Trp residue side-chains were determined. The unit vectors normal to these ring planes were determined (see Supplemental Information for details). For protein anions, we assumed that Asp, Glu carboxylate ions are ionized, and the distance, ro, between a carboxylate oxygen atom and the aromatic ring centers was computed. A distance cutoff |ro| 6 4.5 Å was used to select anion–p pairs. A cutoff for the angle h 6 15° between ro and the normal to the ring was imposed to eliminate anions interacting with the ring edges. For the nucleic acid phosphates, each of the two non-ester oxygen atoms was treated as anion, and for the nucleic acid base ring gravity center only the ring atoms were considered, disregarding the amino and the carbonyl oxygen atoms connected to the nucleobase ring.
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3. Results and discussion 3.1. ‘‘g6’’-Type anion–p geometry Using the distance-angle criteria (see Section 2), we detected 111 ‘‘g6’’-type anion–p interacting pairs in the dataset. With the same criterion, we observed 301 cation–p pairs in the dataset, suggesting that ‘‘g6’’-type anion–p interactions are rarer but unambiguously present in protein structures. For the ‘‘g6’’-type anion– p interactions, the observed distances between pairs were as low as 2.7 Å (Fig. 1b, left) and are comparable to those of the crystal structures of organic salts [6]. B-factors of the ‘‘g6’’-type anion–p interacting atoms were well below 20 (Fig. S1a), suggesting minimal positional ambiguity for these atoms. A distribution of the angle between the carboxylate ion and the ring plane shows that co-planarity is preferred (Fig. 1b, right), possibly to maximize p–p stacking and packing [12]. Even if ‘‘g6’’-type anion–p interactions are considered unfavorable under isolated conditions [5], one can argue that the anion could be held above the ring plane by energetic compensation from other interactions involving the rest of the polypeptide chain. This is difficult to address, due to the need to break down the complete energetic components of the entire folded structure. However, we analyzed the possibility of local polar interactions such as hydrogen bonding or salt-bridges directly involving the anion that register it above the aromatic plane (Fig. 1c). Approximately 70% of the ‘‘g6’’-type anion–p carboxylates are either hydrogen-bonded or are involved in ion-pairs, while the remaining 30% are not, suggesting that this interaction
(a)
pair may not always require compensation. Approximately 60% of the cation–p pairs in the set are hydrogen-bonded or involved in ion-pairs, i.e., even attractively interacting pairs are involved in a network of interactions and the ‘‘g6’’-type anion–p observed here may be exhibiting similar behavior. The depth distribution [13] of the atoms involved in ‘‘g6’’-type anion–p and cation–p pairs (Fig. S1b) suggests that the majority of these are just below the surface, i.e., they are partially buried and the energetics of desolvation discussed earlier [4] for cation–p interactions may similarly be applicable to the ‘‘g6’’-type anion–p interaction. Packing is often a good descriptor of the environment around an atom, as packing is influenced by solvent accessibility, secondary structure and other factors [14]. Using the occluded surface and fraction ray length [15], we computed packing in the neighborhood of this interaction. A smaller fraction ray length indicates better packing, while a longer ray length indicates the opposite. The distribution of the fraction ray length of all Asp + Glu pairs in the dataset is a normal distribution (mean ± S.D. of 0.295 ± 0.049) while that of Asp + Glu pairs involved in ‘‘g6’’-type anion–p is very similar to the that of all Asp + Glu (Fig. 2a, left). This indicates that packing Asp + Glu carboxylates on the ring face does not compromise packing. The same can also be concluded from the packing of aromatic residues involved in ‘‘g6’’-type anion–p (data not shown). As the side-chains of aromatic residues and Asp/Glu tend to pack better than amino acids with aliphatic carbons [14], the formation of ‘‘g6’’-type anion–p interactions will result in well-packed side-chains. In other words, formation of ‘‘g6’’-type anion–p interactions may favorably contribute to packing.
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Fig. 2. Features of ‘‘g6’’-type anion–p in biomolecules: (a) distribution of fraction ray length, a measure of the residue packing density, of carboxylate ions involved in ‘‘g6’’type anion–p (gray bars). This distribution is compared with that of any carboxylate ion (black line) in the protein structure. (b) Inter-residue sequence separation between ‘‘g6’’-type anion–p interacting pairs (left, gray bar) and cation–p (right, black bar). (c) ‘‘g6’’-type anion–p in nucleic acids where the base of the ith nucleotide (purple) pairs with the oxygen atoms of an (i + 2)th phosphate (gray) anion.
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Distribution of the inter-residue separation between the anion and aromatic residues in the primary structure show that ‘‘g6’’-type anion–p interactions occur preferentially between positions that are very close in the sequence, especially between adjacent residues (i, i + 1) or (i, i 1) (Fig. 2b, left). The ‘‘g6’’-type adjacent anion–p pairs are usually in regions with loops and turns (see example Fig. 1c right) and may favorably influence the local folding of polypeptide chains [16] (see text of Fig. S1). 3.2. ‘‘g6’’-Type anion–p in nucleic acids We also examined the existence of ‘‘g6’’-type anion–p in nucleic acids. Similar to proteins, we observed such interactions in nucleic acid loop/hairpin structures (Fig. 2c). In nucleic acid backbones, such as tetraloop hairpins, the base of the ith nucleotide pairs with the oxygen atoms of the (i + 2)th phosphate anion, forming ‘‘g6’’-type anion–p. The distances of the ‘‘g6’’-type anion–p interactions in the nucleic backbone cases are often below 3 Å. While the rest of the phosphate anionic oxygen atoms in the nucleic acid backbone face outward, the ‘‘g6’’-type phosphate anions (poorly solvated) face inward (Fig. 2c). The solvent accessible surface area of inward (OP2 atom) and outward facing phosphate oxygen atoms are 1.5 Å2 and >20 Å2, respectively, suggesting that the nucleic acid base may compensate for the energetics of the desolvation of the anion. In many RNA hairpins, even in structures with low resolutions (P3 Å) (Fig. S2 and associated text), we observed this interaction between the ith base and the (i + 2)th phosphate consistent with the interactions observed in high resolution structures of Escherichia coli 4.5S RNA tetraloops or the hairpin ribozyme stem C loop (Fig. 2c). This suggests that ‘‘g6’’-type anion–p interactions may be a regular feature of nucleic acid loops. In addition, in a few low resolution RNA structures, we also observed ‘‘g6’’-type anion–p between the ith base and the (i 1)th phosphate oxygen anions (Fig. S2), as well as between distant positions in the primary sequence.
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3.3. ‘‘g6’’-Type anion–p and residue conservation We next examined the evolutionary conservation of positions of interacting pairs in proteins using the relative entropy measure (Fig. S3). A similar trend in the relative entropy is observed for both ‘‘g6’’-type anion–p and cation–p. The relative entropies of the anions and the aromatic residues involved in ‘‘g6’’-type anion–p and the respective pairs involved in cation–p seem to be in the intermediate conservation range (Fig. S3) and may suggest that many of these types of non-covalent interactions can be replaced without compromising the stability of the fold. It is important to note that there is a strong co-evolving trend, i.e., a tendency to retain the partner residue when one of the pairing residues is retained in a protein family (Fig. 3a). This trend is similar for both ‘‘g6’’-type anion–p and cation–p. For example, in the Xylanase structures (pfam Glyco_hydro_10 family), the frequency of the catalytic nucleophile Glu, Asp (Glu241 in pdbid 2q8x, Glu236 in 1e0w) in the alignment column is p(E,D) = 0.591. The frequency Glu, Asp (conditional probability) in a subset of sequences of this family that all have either Trp or Tyr or Phe at the alignment position corresponding to the Trp291 in 2q8x or Trp266 in 1e0w, has the probability p(E,D/ WYF) = 0.996 (Fig. 3a and b). The plot of p(E,D) vs p(E,D/WYF) shows that nearly all of the (x, y) points are above the y = x line (Fig. 3a), and the same is true for the plot of p(W,Y,F) vs p(W,Y,F/ ED), suggesting that natural selection favors conservation of such interacting pairs. In the Xylanase example above, structures of a number of enzymes of the family show that in all cases the catalytic nucleophilic carboxylate is held above the aromatic plane, suggesting that a specific orientation (anti to the endocyclic O5– C1 bond [17]) may be necessary for the hydolysis of the substrate glycosidic linkage. Similarly, Lactococcus lactis branched-chain keto acid decarboxylase (KdcA) (pfam TPP_enzyme_N family) catalytic loop residue Asp26 [18] is involved in an ‘‘g6’’-type anion–p interaction with the adjacent Tyr27 with p(D,E) = 0.25 and p(D,E/ WYF) = 0.892 at position Asp26 (2vbf) (Fig. S3 and the associated
(a)
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Fig. 3. Conservation of ‘‘g6’’-type anion–p in biomolecules: (a) plot of p(D,E) vs p(D,E/WYF) (see text) (left) and p(R,K) vs p(R,K/WYF). (b) Sequence logo of a segment of the pfam Glyco_hydro_10 family highlighting the positions involved in ‘‘g6’’-type anion–p (left) and the structure of Xylanase (2q8xB) (right) showing the ‘‘g6’’-type anion–p residues. The secondary structure of this region is shown in blue in Xylanase and is represented below the sequence logo (left) (also see Fig. S3 and associated text).
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text). Some of the examples of co-evolving sites observed here include enzyme catalytic sites. This may suggest that the supporting role of the ‘‘g6’’-type anion–p interaction is in orienting catalytic residues, and could be useful in the design of artificial enzymes that require grafting catalytic residues onto a stable protein scaffold [19]. 3.4. ‘‘g6’’-Type anion–p at interfaces Though no extensive analysis of interfaces is carried out here, we discuss a few interfaces, specifically those involving RNA-protein interactions corresponding to crucial biological processes (Fig. 4). The RRM (RNA recognition motif) of the U1A protein binds to U1 snRNA hairpin II. The structure of the complex reveals that RRM Asp92 stacks in parallel on the Cyt12 residue of the hairpin loop, [20] forming an ‘‘g6’’-type anion–p. The same feature is observed in the structure of the RRM of U2B’’ bound to hairpin IV of U2 snRNP (Fig. 4). U2B’’ Asp92 stacks on Guanine12 of the hairpin loop of U2 and this arrangement seems to be a conserved feature of spliceosomal RNA hairpins involving a continuous stack of four planes of Phe56, A11, C/G12 and Asp92-carboxylates, in which the two amino acid side chains appear to hold the interior stacked bases like clamps at either end [21]. Next we discuss the interaction between tRNA and two distinctly different b-barrel domains. These barrels are the elongation factor isomerase (EI) type of b-barrel in EF-Tu (elongation factor Tu) and the L25-like barrel in amino acyl-tRNA synthetase (aaRS) that binds tRNA. EF-Tu, in constrast to
aaRS, forms complexes with all aa-tRNAs. Hence, EF-Tu recognizes common features of tRNA including the 50 CCA-OH acceptor arm [22]. The terminal 50 -A of the Phe-tRNAPhe acceptor arm stacks against Glu271 of the center b-barrel domain of Thermus acquaticus EF-Tu (Fig. 4). This Glu residue is conserved in the sequences of Homo sapiens, Saccharomyces cerevisiae, E. coli, Bacillus subtilis, Sulfolobus solfataricus, Haloarcula marismortui, and T. aquaticus EF-Tu [23]. This interaction is also observed in the structure of E. coli Cys-tRNACys complexed to EF-Tu [23]. The b-barrel domain of Gln-RS specifically recognizes the anticodon loop of Gln-tRNAGln, enabling Gln-RS to discriminate between cognate and non-cognate tRNAs [24]. The three Gln-tRNA anticodon bases (CUG) are aligned in different directions to fit into base-specific pockets [24]. Glu519 stacks onto U35 in the U35 base-specific pocket. The U35 pocket is thought to be the tightest among the three pockets [24], and packing of the carboxylate ion onto the U35 face may be equally important to the interpretation of the genetic code. A BLAST search (20 iterations, e value cutoff 60.001 and alignment coverage P60%) with the sequence of this domain shows that more that 60% of the retrieved sequences have Glu/Asp residues corresponding to the position of Glu519 in Gln-RS, suggesting strong preference for the anion in the U35 pocket. Other examples of interfacial ‘‘g6’’-type anion–p are provided in the Supplemental Information (Fig. S4). Aromatic residues have long been known for their functional versatility (Fig. S6). With the examples described above, it is clear that the number of versatile functions for aromatic residues in
Fig. 4. ‘‘g6’’-Type anion–p in interfaces: RRM (split-pea green, 1a9n) of U2B00 (spliceosomal machinary) in complex with U2 snRNA (green, top left), EF_TU b-barrel domain (split-pea green, 1b23) in complex with the acceptor arm of Phe-tRNAPhe (green, top right), Glutamyl tRNA synthetase b-barrel domain (split-pea green, 1gtr) in complex with the anticodon arm of Gln-tRNAGln (green, bottom left), Syntenin tandem PDZ (green, 1obx) in complex with IL5Ra (orange, bottom right) (also see Fig. S4 and associated text).
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macromolecules is increasing. However, in the absence of point mutation studies, the energetics of the ‘‘g6’’-type anion–p interaction remain to be studied in the future, in a manner similar to experiments performed with cation–p interactions in globular proteins [25]. It is encouraging to note that in a number of examples the protein sequence positions involved in ‘‘g6’’-type anion–p are not only conserved, but the interacting pairs also show co-evolving trends suggesting a possible energetic or functional coupling between these positions [26]. This co-evolution is similar to that of cation–p interactions, the energetics of which are detailed by isostearic point mutations in globular proteins that are suggested, on average, to have stabilizing effects [25]. Similarly, one may expect to find that ‘‘g6’’-type anion–p interactions have stabilizing effects on average, although these interactions may be very weak. Lastly, it is interesting to note that even in the absence of highly electron-withdrawing groups in the aromatic ring we observe ‘‘g6’’type anion–p in macromolecules, even contributing to crucial biomolecular recognition. This suggests that we still lack a comprehensive picture of the nature of weak molecular interactions, and this study provides an impetus for the development of appropriate chemistry theory to accommodate these weak molecular interactions and their application in molecular design.
[10]
Acknowledgements
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The study was supported by the SDSU Agricultural Experimentation grant 3AH386 and Biological Control and Analysis by Applied Photonics (BCAAP), as well as the South Dakota 2010 center grant 3SG163 to S.C. The authors thank Adam Hoppe for carefully reading the manuscript and providing helpful suggestions. The authors thank Jay Shore for providing helpful suggestions.
[17]
[6]
[7]
[8]
[9]
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[12] [13] [14]
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[19]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012.10. 017. References [1] Kim, K.S., Tarakeshwar, P. and Lee, J.Y. (2000) Molecular clusters of pi-systems: theoretical studies of structures spectra, and origin of interaction energies. Chem. Rev. 100, 4145–4186. [2] Jain, A., Ramanathan, V. and Sankararamakrishnan, R. (2009) Lone pair pi interactions between water oxygens and aromatic residues: quantum chemical studies based on high-resolution protein structures and model compounds. Protein Sci. (Protein Society) 18, 595–605. [3] Dougherty, D.A. (1996) Cation–pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271, 163–168. [4] Gallivan, J.P. and Dougherty, D.A. (2000) A computational study of cationp interactions vs salt bridges in aqueous media: implications for protein engineering. J. Am. Chem. Soc. 122, 870–874. [5] Philip, V., Harris, J., Adams, R., Nguyen, D., Spiers, J., Baudry, J., Howell, E.E. and Hinde, R.J. (2011) A survey of aspartate-phenylalanine and glutamate-
[20]
[21]
[22]
[23]
[24] [25]
[26]
4185
phenylalanine interactions in the protein data bank: searching for anion–pi pairs. Biochemistry 50, 2939–2950. Albrecht, M., Wessel, C., de Groot, M., Rissanen, K. and Luchow, A. (2008) Structural versatility of anion–pi interactions in halide salts with pentafluorophenyl substituted cations. J. Am. Chem. Soc. 130, 4600–4601. Dawson, R.E., Hennig, A., Weimann, D.P., Emery, D., Ravikumar, V., Montenegro, J., Takeuchi, T., Gabutti, S., Mayor, M., Mareda, J., Schalley, C.A. and Matile, S. (2010) Experimental evidence for the functional relevance of anion–pi interactions. Nat. Chem. 2, 533–538. Escudero, D., Frontera, A., Quinonero, D. and Deya, P.M. (2009) Interplay between anion–pi and hydrogen bonding interactions. J. Comput. Chem. 30, 75–82. Wang, G. and Dunbrack Jr., R.L. (2003) PISCES: a protein sequence culling server. Bioinformatics 19, 1589–1591. Punta, M., Coggill, P.C., Eberhardt, R.Y., Mistry, J., Tate, J., Boursnell, C., Pang, N., Forslund, K., Ceric, G., Clements, J., Heger, A., Holm, L., Sonnhammer, E.L., Eddy, S.R., Bateman, A. and Finn, R.D. (2012) The Pfam protein families database. Nucleic Acids Res. 40, D290–301. Li, W. and Godzik, A. (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659. Marsili, S., Chelli, R., Schettino, V. and Procacci, P. (2008) Thermodynamics of stacking interactions in proteins. Phys. Chem. Chem. Phys. 10, 2673–2685. Chakravarty, S. and Varadarajan, R. (1999) Residue depth: a novel parameter for the analysis of protein structure and stability. Structure 7, 723–732. Fleming, P.J. and Richards, F.M. (2000) Protein packing: dependence on protein size, secondary structure and amino acid composition. J. Mol. Biol. 299, 487– 498. Pattabiraman, N., Ward, K.B. and Fleming, P.J. (1995) Occluded molecular surface: analysis of protein packing. J. Mol. Recognit. 8, 334–344. Baldwin, R.L. and Rose, G.D. (1999) Is protein folding hierarchic? II. Folding intermediates and transition states (vol. 24, p. 77, 1999). Trends Biochem. Sci. 24, 185–186. Heightman, T.D. and Vasella, A.T. (1999) Recent insights into inhibition, structure, and mechanism of configuration-retaining glycosidases. Angew. Chem., Int. Ed. 38, 750–770. Berthold, C.L., Gocke, D., Wood, D., Leeper, F.J., Pohl, M. and Schneider, G. (2007) Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction. Acta Crystallogr., Sect. D: Biol. Crystallogr. 63, 1217–1224. Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Rothlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L. and Baker, D. (2008) De novo computational design of retroaldol enzymes. Science 319, 1387–1391. Oubridge, C., Ito, N., Evans, P.R., Teo, C.H. and Nagai, K. (1994) Crystal-structure at 1.92 Angstrom resolution of the RNA-binding domain of the U1a spliceosomal protein complexed with an RNA hairpin. Nature 372, 432–438. Price, S.R., Evens, P.R. and Nagai, K. (1998) Crystal structure of the spliceosomal U2B00 –U2A0 protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650. Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F.C. and Nyborg, J. (1995) Crystal-structure of the ternary complex of PheTrna(Phe), Ef-Tu, and a Gtp Analog. Science 270, 1464–1472. Nissen, P., Thirup, S., Kjeldgaard, M. and Nyborg, J. (1999) The crystal structure of Cys-tRNA(Cys)-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA. Struct. Fold. Des. 7, 143–156. Rould, M.A., Perona, J.J. and Steitz, T.A. (1991) Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature 352, 213–218. Prajapati, R.S., Sirajuddin, M., Durani, V., Sreeramulu, S. and Varadarajan, R. (2006) Contribution of cation–pi interactions to protein stability. Biochemistry 45, 15000–15010. Lockless, S.W. and Ranganathan, R. (1999) Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299.