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The Aromatic Stacking Interactions Between Proteins and their Macromolecular Ligands Mohammad Mizanur Rahman1,2, Ziyad Tariq Muhseen1, Muhammad Junaid1 and Houjin Zhang1,* 1

Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, 1037 Luoyu Rd., Wuhan 430074, Hubei, China; 2Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh Abstract: Aromatic stacking interactions arise from the attractive force between the π-electron clouds in the neighboring aromatic groups. The aromatic stacking is common between proteins and small molecules. The stacking interactions at the interfaces of proteins and other macromolecules are relatively rare. However it contributes to a significant portion of the stabilizing forces. In the proteinprotein complexes, aromatic interactions are involved in the protein oligomerization, such as dimer, trimer and tetramer formation. Also, aromatic residues can bind to nanoparticles through stacking interactions which offer them stronger affinity than other residues. These interactions play crucial roles in proteinnanoparticle conjugation. In the protein-nucleotide complexes, the specific recognitions are realized through stacking interactions between aromatic residues and the bases in the nucleotides. Many nucleoproteins use aromatic stacking to recognize binding site on DNA or RNA. Stacking interactions are involved in the process of mismatch repair, strand separation, deadenylation, degradation and RNA cap binding. They are proved to be important for the stability of complexes. The aromatic stacking is also the underlying reasons of many fatal diseases such as Alzheimer, cancer and cardiovascular diseases. The chemicals that can block the stacking interactions could have potential pharmaceutical values. In this review, we summarize recent finding regarding the functions of aromatic stacking interactions in the protein-macromolecule complexes. Our aim is to understand the mechanisms underlying the stacking-mediated complex formation and facilitate the development of drugs and other bio-products.

Keywords: Aromatic amino acid, carbon nanotube, π-π stacking, protein-nucleotide complexes, protein-nanoparticle interaction, protein-protein interaction. 1. INTRODUCTION Aromatic compounds contain cyclic aromatic rings that are unsaturated and planar [1]. The stability of these compounds results from the π -electrons situated above and below the plane of the aromatic ring. Together, these electrons form a π-electron cloud surrounding the ring. The flat face of an aromatic ring has a partial negative charge because of these π electrons. Aromaticity is a chemical term that refers to the characteristics associated with the cyclic and planar aromatic system. It can be regarded as a manifestation of cyclic delocalization and resonance. The well-studied model system of aromaticity is the benzene molecule [2]. The aromatic rings are present in the side-chains of three amino acids with 6-member ring: phenylalanine (F), tyrosine (Y), tryptophan (W). And aromaticity is also found in the 5member ring residue, such as histidine (H). These amino acids are called aromatic amino acids [3]. The aromatic rings of these residues interact with other aromatic systems by

*Address correspondence to this author at the Department of Biotechnology, Faculty of Life Science and Technology, Huazhong University of Science and Technology, Zipcode 430074, Wuhan, Hubei, China; Tel: +86 02787793085; Fax: +86 27 87793085; E-mail: [email protected]

attractive non-covalent force [4]. Such interactions are called aromatic stacking, π -stacking or aromatic-aromatic interactions. The common features of aromatic-aromatic interactions include (i) nonpolar interactions (ii) the distances of two phenyl rings fall between 4.5 Å to 7 Å, (iii) the dihedral angles range from 30° to 90° (iv) a typical non-bond energy of-1 to -2 kilocalories per mole [5]. Stacking between different aromatic systems is a prominent stabilizing force in the biomacromolecules and their related complexes [6]. Specifically, the aromatic residues in proteins play significant roles in the stability of interacting complex, in substrate binding as well as drug designs [7]. The main factors that affect the strength of aromatic stacking interactions are the geometry and proper orientations of interaction molecules [8]. A study of 2280 α-helices in 434 protein chains has observed that the geometry of the stacking system depends on the sequence between the interacting partners [9]. The study also demonstrated that His is the most conspicuous residue in the context of helix stability. Similarly, Samanta and coworkers analyzed the geometry of the aromatic side chains interactions involving Trp indole ring [10]. The Phe ring prefers either offset-stacked arrangement or perpendicular orientation with Trp by edge-to-face stacking, while Tyr prefers

face-to-edge or face-to-face fashion with Trp. Trp-Trp interaction prefers edge-to-face style. On the other hand, histidine preferentially stacks with Trp by parallel stacking. The energetically favorable stacking might come from the positively charged His imidazole ring under physiological conditions. As suggested by the mutagenesis assay, this charged His-Trp stacking can contribute to the protein stability by more than 4 kJmol-1 [11]. The aromatic stacking not only provides the stability and mechanical insight of proteins and protein/ligand complexes, but could also be implemented in the protein design process. The judicious placements of aromatic residues and the resulting geometric parameters could be useful tools in de novo protein design and might be useful for modeling protein interactions [12]. Additionally, aromatic residues can preferentially bind with nanoparticles to form protein-nanoparticle hybrid that has applications in many fields of science such as biomedicine, catalysis, and water treatment [13]. Based on ππ stacking interactions, some nanoparticles have already been developed such as carbon nanotubes (CNTs) [14], fullerene [15], graphene [16]. Furthermore, aromatic amino acids are key residues that interact with DNA or RNA, which provide recognition sites for the ligands to bind [17]. These aromatic residues play important roles in gene expression, damaged DNA repair, RNA cap binding [18]. The mutations on these aromatic residues are often found to be the underlying reasons for diseases caused by faulted protein-nucleotide recognition [19]. Although the π-π aromatic stacking in protein-ligand interactions has important physiological roles in organism, no review paper has covered this subject yet. Due to the abundance of stacking interactions between protein and small molecules, the current review will focus on the stacking interactions between proteins and macromolecular ligands. 2. AROMATIC INTERACTIONS PROTEIN COMPLEXES

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Similarly, stabilization of the dimer interface in UDPglucuronosyltransferase 2B7 is achieved by Phe187/Phe187 and Phe189/Phe189 aromatic stacking interactions along with two salt bridges (H183/E200) and two S-aromatic (face) interactions (F187/M198) [24]. The π -π stacking on the dimer interface is also observed in farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). Crystal structures of Thermotoga maritima OPPs indicate Phe117 form a π -π stacking with Phe117 from the other chain in the dimer [25]. Likewise, in the structures of Escherichia coli FPPs, Phe-122 residues from the opposing chains stack on each other to connect the two protomers [26]. Also, the C-terminal domain (CTD) of HIV-1 integrase form dimer through stacking interaction between Trp243 on neighboring chain [27]. The structure of Trypanosoma brucei ornithine decarboxylase (TbODC) forms a twofold symmetric dimer. The TbODC monomer consists of two domains, namely, N-terminal domain and C-terminal domain. The two monomers complex with each other through aromatic interactions. The interface is formed by the interaction between N-terminal domain and C-terminal domain. A prominent feature at the dimer interface is the aromatic amino acid zipper (Fig. 1). This zipper structure plays a key role in dimerization as well as positioning pyridoxal 5’phosphate for catalysis [28]. Likewise, the phage λ cI repressor also forms dimer to distinguish DNA sequence specificity in operator [29]. The side chains of two symmetrically related Tyr88, although far from the DNA -binding site, stack with face-to-face at the dimer interface, which is vital for the dimer stability as well as the operator recognition.

PROTEIN-

It has been long known that the aromatic interactions contribute to the overall stability of protein molecules [20]. Recently, studies have demonstrated that the aromatic residues are also the stabilizing factors for the protein-protein complexes. The aromatic stacking interactions are formed between neighbouring proteins in the complexes and, therefore, provide the stabilizing forces. A good example of such interaction can be found in the glutathione transferases (GSTs) dimer interfaces [21]. On the surface of the GST from malaria vector Anopheles dirus, the Phe104 is exposed and forms a stacking interaction with Phe104 on the other protomer. Such interaction is strengthened by the nearby hydrophobic residues (Val107 and Leu103). These residues altogether form a lock-and-key motif on the dimer interface. This motif is not uncommon in other GST classes. In human GSTP1-1 (GST class Pi), the Try47 protrudes into the hydrophobic pocket formed by the Val90, Phe127, Leu130 from the neighbouring chain [22]. In the alpha-class GST hGSTA1-1, a lock-and-key motif is found at Phe52 and the residues surrounding it [23]. These motifs are proven to be important to the function of GSTs. The mutations on these residues diminish the enzyme activities, suggesting the importance of aromatic stacking in the function of GSTs.

Fig. (1). Representative diagram of aromatic stacking in dimer interfaces. The T. brucei ornithine decarboxylase (TbODC) dimer interface is stabilized by aromatic stacking between Tyr323Phe397, Tyr323'-Phe397', Tyr323-Tyr331', Tyr331-Tyr323'. The different monomers are colored as black or gray.

In addition, many signaling molecules contains modular domains which are responsible for protein-protein interactions [30]. The SH3 domains of epidermal growth factor receptor pathway substrate 8(Eps8) protein form an intertwined, domain-swapped dimer. The dimer interface is stabilized by aromatic stacking of Trp40 and Phe52 with their counterparts in the other monomer [31]. Also, the receptor tyrosine kinases are involved in lateral dimerization where aromatic residues are important in specific transmembrane

(TM) helix-helix interaction [32]. NMR structure revealed that the EphA2tm dimer is formed by intermolecular aromatic stacking between the side chains of two Phe556 and two Phe557 which plays key roles in TM helix packing [33]. Not only are the aromatic stacking interactions found in the dimers, they are also found in other protein complex forms, such as trimers and tetramers. The microsomal prostaglandin E synthase 1 (mPGES-1) is a homo-trimer with inter-helical interactions within each monomer formed by aromatic stacking [34]. A three-layered π - π stacking formed by helix 2 Phe82 (H2), helix 3 Phe103 and Phe106. The strong aromatic stacking is the main driving force for mPGES-1 subunit stability. Recently, Wang et al. found aromatic stacking at the interface of ketoreductase SiaM protein tetramer [35]. Phe123 and Tyr111 form a T-shaped aromatic stacking in the N- terminal interface, while Phe227 of one protomer form a parallel-displaced π-π stacking interaction with Phe227 of another protomer in the C-terminal interface (Fig. 2). In addition, Tyr235 inserts into the binding pocket in the opposite protomer and form a N-H/Pi interaction with an arginine residue nearby. Subsequent mutagenesis study confirmed the importance of aromatic stacking for maintaining the structural integrity and enzymatic activity of SiaM.

achieved through aromatic stacking between tryptophan and the aromatic residues in the antibody [37]. In addition, the aromatic stacking is found at the recognition sites of three human antibodies against influenza virus hemagglutinin (HA). Since the HA mediates the receptor binding and viral entry during influenza infection, such binding pattern could well serve as a guide for drug design [38]. AROMATIC STACKING 3. NANOPARTICLE COMPLEXES

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PROTEIN-

The aromatic stacking interactions play a significant role in the formation of protein-nanoparticle conjugate. The surface chemistry of CNTs and proteins includes several kinds of interactions such as π-π stacking, hydrophobic, surfactantlike and charge π interactions [39]. The aromatic residues of proteins are the main binding sites for CNTs. The side chains of these residues form strong π-π stacking with the surface of the CNT, thus, therefore, provides the main driving force for the protein/CNT binding [40]. The π−π stacking interactions of proteins with CNTs appear to be of substantial importance because of extended π conjugation on CNTs [41]. It has been reported that the adsorption of proteins into CNTs depends on the content of aromatic residues in the protein sequence. Proteins with many aromatic residues bind to CNTs easily through π -π stacking interactions [42]. Numerous studies indicate that aromatic residues bind to CNTs more sturdily than other amino acids. Among aromatic residues, Trp holds the highest affinity to CNTs, which is followed by Tyr and Phe [39, 43]. A study by Ge et al. demonstrated that human serum proteins interact with SWCNTs by a competitive binding pattern with different packing and adsorption capacity [44]. The competitive bindings of proteins-SWCNT significantly alter the pathways of cellular interaction. This alteration reduces cytotoxicity of protein-coated SWCNTs. It was also reported that the SWCNT can inhibit amyloid formation. The ring of single wall carbon nanotube (SWCNT) forms a parallel π stacking with the aromatic ring of F19 and indirectly inhibits the possible cross-strand stacking in Aβ assembly (Fig. 3) [45].

Fig. (2). The aromatic stacking in the interfaces of SiaM. The ketoreductase SiaM forms a tetramer in solution. Aromatic stacking interactions are found in both N-terminal and C-terminal interfaces. The Phe227 and Phe227' form a parallel aromatic stacking interaction (shown in box). The monomers are colored in black or gray. The aromatic residues are shown in sticks and the rest of the molecules are shown in ribbon.

A bioinformatics analysis of antibody/antigen complex in PDB database indicates that aromatic stacking is an important mechanism for antigen recognition. The aromatic residues in the antigens serve as the epitopes. The antibodies bind these epitopes with a deep cavity formed between L3 and H3 regions [36]. A good example can be found in the binding of PLY-5, a mouse monoclonal antibody, which recognizes the conserved undecapeptide tryptophan-rich loop (ECTGLAWEWWR) of bacterial cholesterol-dependent cytolysins (CDCs). It is predicted that the tryptophan-rich loop is exposed on the surface of CDCs and serve as the recognition site for antibody. The antibody/antigen binding is

4. AROMATIC INTERACTIONS NUCLEOTIDE COMPLEXES

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4.1. Protein-DNA Interactions Protein-DNA interactions are essential for all organisms to fulfill their functions [46]. Over the years, many efforts have been made to understand the binding of protein and DNA at atomic level. In the B-form DNA structure, the bases are shielded by the DNA backbones and not accessible from the outside. The DNA-protein aromatic stacking is often found in the places where the normal DNA structure is altered. Cleavage of the N-glycosidic bond is the most common lesion under physiological conditions and leaves a site without a base [47]. A recent study shows that, in KlenTaq, the Tyr671 protrudes into the DNA lesion through aromatic stacking with the base and guide the incoming NTP to repair the abasic site. In human alkyladenine DNA glycosylase, the key tyrosine residues can be substituted with tryptophan to initiate the base excision repair pathway, indicating the aromaticity is the key for its function [48]. Furthermore, the side

Fig. (3). The schematic representation of proposed inhibitory mechanism of Aβ (16-22) nucleation by SWCNT. The random coil amyloid chains aggregate through aromatic stacking (shown in box) and form beta-sheet-rich oligomers. SWCNT may block the self-assembly process by stacking on the aromatic residues and lead to the formation of disordered coil aggregate.

chains of aromatic residues of base flipping enzymes can form an aromatic stacking with flipped base in DNA [49]. The E. coli AlkA protein flips a 1-azaribose abasic nucleotide out of DNA by stacking of Trp272 side chain with alkylated bases [50]. Another study also suggested that Trp272 is important for substrate recognition and repair system of base-excision DNA repair proteins [51]. These results suggest prominent roles of aromatic stacking in damaged DNA recognition. A notable example of aromatic stacking is also observed in prokaryotic cold shock proteins (Csp) family [52]. Csp proteins stimulate the transcription of cold shock inducible genes and often initiate translation at low temperature by destabilizing non-productive secondary structures of nucleic acids [53]. Six highly conserved aromatic residues (Phe15, Phe17, Phe27, Phe30, Trp8, and His29) are found in a patch and they form the nucleic acid binding site [54]. Substitutions of these aromatic residues to non-aromatic residues abolish DNA binding affinity of Bacillus subtilis CspB [55]. E. coli CspA is a β-sheet protein of only 69 amino acids and it possesses a continuous nonpolar patch. It has been demonstrated that the aromatic cluster is required not only for protein function but also for protein stability [56]. Phadtare et al. observed that the mutations on the aromatic patch (Trp10, Phe17, Phe19, Phe30, His32, and Phe33) of E. coli CspE have no effect on RNA binding. Instead, the aromatic residues (Phe17, Phe30 and His32) are critical for DNA melting activity, cold acclimation of cells and transcription antiter-

mination [57]. A recent study also found the importance of aromatic stacking in protein-oligonucleotide interaction by analyzing crystal structure of Csp from Bacillus caldolyticus (Bc-Csp) with dT6 oligonucleotide [58]. The oligonucleotide-Bc-Csp interaction is dominated by aromatic stacking between the side chains of Trp8- T6, Phe17- T5 and Phe27T4 residues. Such aromatic stacking plays prominent roles for Bc-Csp’s function as RNA chaperones. Aromatic interactions also have a key role in DNA strand separation. Dda, a phage T4 helicase, plays a pivotal role in strand separation [59]. It was suggested that helicase translocation along single-stranded DNA (ssDNA) and DNA strand separation is coupled. X-ray crystallography of the DdassDNA binary complex reveals a domain called “pin”. Structural analysis of “pin” shows a Phe residue at active site that mediates a transient base-stacking interaction for the separation of dsDNA [60]. Besides, it has also been suggested that, Dda tightly couples translocation of helicase to strand separation site. The prominent roles of aromatic residues in strand separation have also been observed in a study by Kim JW et al. [61]. X-ray crystal structure of the HCV nonstructural protein 3 (NS3) helicase with (dU)8 oligonucleotide shows that Trp501 is involved in a base stacking interaction crucial for helicase activity. Mutations of the Trp501 to nonaromatic amino acids lead to a damage in RNA unwinding activity, although for DNA the activity remains unchanged. Molecular analysis of the enzyme-substrate complex reveals a π-facial hydrogen bond interaction between the Trp aro-

matic ring and ribose 2’-OH. Furthermore, this site was mutated to other aromatic residues. Interestingly, they found that the mutations fully restore the damaged RNA unwinding activity, indicating the crucial role of the aromatic ring for the RNA helicase function. Moreover, Yoshikawa and coworkers observed a specific role of aromatic residues in the recognition of Holliday junction in E. coli [62]. E. coli RuvC resolvase is an endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and repair. Structural analysis by X-ray crystallography suggests that a Phe residue (Phe69) faces the catalytic center of the enzyme. In the interaction of RuvC with nucleotides, the aromatic ring of Phe69 contributes to the DNA binding by stacking with a nucleotide base, which leads to the disruption of base paring in the Holliday junction. Substitutions of Phe69 with other amino acids abolish the ability to cleave Holliday junction. The aromatic stacking is involved in the process of DNA mismatch repair. Several studies have shown close contact between aromatic residues and bases of mismatched DNA in E. coli [63]. The bacterial MutS protein binds with mismatch DNA. Two subunits namely, subunits A and B of the MutS homodimer, have distinctive roles in the binding. Drotschmann K and coworkers demonstrated that Phe337 and Glu339 of MutS are responsible for mismatched basespecific interactions [64]. But residues of both subunits asymmetrically contact the DNA backbone surrounding the mismatched base. This result is confirmed by a recent study that a Phe is stacked against thymine in G:T mismatch-bound MutS [65]. Similarly, a short patch repair protein Vsr from E. coli recognizes a TG mismatched base pair in damaged DNA [66]. Crystal structure of Vsr complexed with DNA reveals that Phe67, Trp68, and Trp86 intercalate between the TG mismatch and the adjacent AT base pair [67]. The side chain of Phe67 is stacked with the central AT base pair in the recognition sequence while Trp68 side chain stacks with the mispaired thymidine. Trp86 stacked with the two sequential sugar rings of the strand opposite the cleaved strand. In ssDNA, the bases are exposed and may form aromatic stacking with binding proteins. It was shown that, in E. coli single-strand DNA binding protein (EcoSSB), Trp54 stacks on the nucleotide bases [68]. Similar stacking interactions between single-strand DNA and its binding proteins are also found in eukaryotes [69]. In the ssDNAbinding domain of human replication protein A (RPA), a series of Phe residues (F238, F269, F386) are involved in the aromatic stacking with DNA bases. The heterotrimeric (ssDNA)-binding protein, RPA, contains four ssDNA binding domains within its subunits [70]. Two conserved aromatic residues of each domain contribute in high affinity binding of RPA with ssDNA. A rare case of stacking interaction can be found in the ssDNA-antibody complex [71]. Anti-DNA antibodies that bind to hairpin-forming DNA ligands are particularly important in the pathogenesis of autoimmune disease systemic lupus erythematosus (SLE) [72]. A recombinant anti-ssDNA Fab (DNA-1) binds with oligonucleotides by inserting thymine bases between Tyr residues (Fig. 4). The critical Tyr residues involved in the aromatic stacking interactions are located at positions 100 and 101 [73].

Fig. (4). An unusual aromatic stacking in anti-ssDNA Faboligonucleotide complex. Two aromatic residues, Tyr 100 and Tyr 101, sandwich thymine base and form a continuous stacking, which is thought to contribute to the stability of the complex. The sandwich stacking is shown in circle. The ssDNA is shown in black and the Fab is in gray.

4.2. Protein-RNA Interactions Protein-RNA complexes have important roles in many biological processes, including mRNA stabilization, translation, and ribosome formation [74]. It is well established that DNA and RNA behave differently with amino acids [75]. Most protein-DNA interactions involve phosphate atoms, while protein-RNA interactions frequently utilize ribose atoms and bases. As a result, aromatic residues take part in the ligand recognition of many RNA-recognizing proteins through stacking interactions [76]. These RNA-binding proteins have RNA recognition motif (RRM) which is also called RNA nucleoprotein (RNP) domain. The RRM is a ubiquitous RNA binding domain of 90-100 amino residues that form an antiparallel β-sheet held by a pair of alpha helices [77]. Different RRMs modify the basic structure with high specificity to recognize the diverse single-stranded RNA targets. Birney et al. found three highly conserved aromatic residues in RRM that interact with RNA [78]. Structural studies of many RRM-RNA complexes have demonstrated that aromatic residues of RRM form stacking interaction with nucleobase for base recognition and specific binding [79]. U1A is a spliceosome protein of U1 snRNP that contains two RRMs. In these two RRMs, only the Nterminal RRM domain can binds with RNA. U1A binds with stem-loop 2 of U1 snRNP and splice most eukaryotic premRNA. Two highly conserved aromatic residues in RRM, Tyr 13 and Phe56, are found in U1A. Several studies observed that Tyr 13 stacks with base C5 while Phe56 stacks with A6 base in the untranslated region (UTR) (Fig. 5) [79a, 80]. Nolan and coworkers substituted Phe56 with other aromatic residues, Leu, and Ala to assess the contribution of Phe56 to the stability of the U1A N-terminal RRM and stemloop 2 complex [81]. Replacements of Phe56 with other aromatic residues do not destabilize the complex. However, substitution of Phe56 with Ala and Leu resulted in a binding energy loss of 5.5 and 4 kcal/mol respectively. These results strongly suggest the importance of an aromatic residue at

position 56 for high affinity binding of U1A to RNA. Recently, Shiels et al. observed that substitution of Phe56 with Tyr or Trp increased the rate of complex dissociation, while substitution with His decreased the rate of association of the complex, implying an alteration in the initial complex formation [82]. Furthermore, substitution of Phe56 with Leu reduced U1A ability to correctly recognize stem-loop 2. These experiments suggest that Phe56 contributes to high affinity binding by stacking with base A6. Besides, Phe56 also participate in energetically coupled interactions networks that enables the conserved aromatic residue to play a significant role in recognizing target site.

Fig. (5). Stacking in the human U1A spliceosomal protein-RNA complex. The N-terminal RRM domain interacts with RNA by aromatic stacking. Two highly conserved aromatic residues, Tyr13 and Phe56, stack with base C5 and A6 respectively (shown in box), which plays an important role in the recognition of RNA molecules. In the box, the RNA molecule is colored in gray and the protein molecule is in black.

L5 is an example of ribosomal protein binding to eukaryotic 5S rRNA. The L5 is in complex with 5S rRNA and then stored in the cytoplasm in Xenopus. 5S rRNA binds to either the related zinc finger protein, p43 or transcription factor IIIA (TFIIIA) during the primary phases of oogenesis [83]. The L5 contains many aromatic residues, including 19 tyrosines that interact with RNA [84]. A study by DiNitto and coworkers showed the role of numerous conserved aromatic residues in the binding of L5 protein to 5S rRNA [85]. By comparative sequence analysis, some conserved aromatic residues, mostly tyrosines, were identified. Substitutions of Tyr86, Tyr99, or Tyr226 with alanine abolish RNA-binding activity. However, substitution with Phe at Tyr86 and Tyr226 does not change binding affinity. These results indicate that intermolecular aromatic π -stacking involving two Tyr residues, Tyr86 and Tyr226, are important for specific binding and the L5-5S rRNA complex formation. In addition, several aromatic stacking interactions are also found in the structures of other RNA-RNP complexes such as MS2 viral coat protein-operator RNA [86], anticodon interactions with tRNA synthetases [87], ribosomal protein L30autoregulatory RNA element [88] and λ N protein-boxB RNA etc [89].

Aromatic stacking is also participated in the mRNA deadenylation and degradation process by coupling TIS11d protein to the 3′untranslated region (3′UTR) class II AU-rich element (ARE). Aromatic residues in the tandem zinc finger (TZF) domain of the TIS11d recognize ARE and further stabilize the complex [90]. The TIS11d proteins contain a highly conserved TZF domain with several aromatic residues and two CCCH zinc finger motifs including a (R/K)YKTEL motif for the mRNA-binding activity. Substitution of the conserved aromatic residues abolishes the TZF domain’s RNA-binding activity [91]. Hudson et al. demonstrated that the interface between the TZF domain and the 5′UAUUUAUU-3′ ARE motif is dominated by several aromatic π stacking along with some hydrogen-bonding interactions [92]. The side chains of Tyr170 of finger 1 form a coplanar stacking with the bases of U8 and U9. Another aromatic amino acid, Phe176 intercalates between U6 and A7 bases. Furthermore, Tyr208 of finger 2 intercalates between U4 and U5 with its aromatic side ring while Phe214 stacked between U2 and A3. The intercalated aromatic residues are crucial for high-affinity binding of TZF with ARE. The continuous stacking of aromatic bases is usually found in the helical nucleotide structures. However, the sandwiched stacking between residues and bases, like those in the TZF/RNA complexes, can be found in other proteinnucleotide complexes. The mammalian RNA-binding protein, Musashi1 (Msi1), regulates the target mRNAs translation by unique stacking [93]. The N-terminal regions of mouse Msi1 and Msi2 contain two RNA binding domains (RBD1 and RBD2) [94]. The complex structure of Msi1 RBD1: r (GUAGU) shows that the adenine residues at third and guanine at the fourth positions are stacked on the conserved Phe23 and Phe65, respectively. Also an unusual sandwiched stacking is formed by stacking between Phe96, the third adenine and Phe23. Additionally, the nuclear cap-binding complex (CBC) specifically recognizes and binds to mRNA 5’ cap. This binding is crucial for a vast majority of mRNA metabolic events such as pre-mRNA splicing and U snRNA transport. It is also proved to be important for the protection of mRNA from degradation. The CBC-RNA interaction is mediated by several hydrogen bonds and the aromatic stacking of the Tyr side chains with the first two bases of the capped mRNA [95]. In addition, Worch et al. described the diverse role of Tyr in the human nuclear cap binding complex [96]. It appears that the Tyr residues stabilize the cap binding through stacking interactions. Likewise, in Trypanosoma brucei cap 2' RNA methyltransferase, two aromatic amino acids, Tyr18 and Tyr187 stack with the guanine ring of the cap [97]. The removal of these aromatic residues abolishes cap-specific RNA-binding activity. 5. AROMATIC STACKING AND PATHOGENESIS OF DISEASES The aromatic stacking is a contributing factor for many severe diseases. The nucleotide excision repair (NER) is an important step in DNA repair process, and it is found in almost all eukaryotes [98]. Thus, any defect in the NER system can cause serious disorders in human body such as Cockayne syndrome, Xeroderma pigmentosum and trichothiodystrophy [99]. Several proteins can carry out in

Fig. (6). The 3D representation of the XPF homodimer. The monomers are colored in black or gray. The aromatic stacking interactions are formed between the Phe168 and Phe119 of one monomer and Phe19 and Phe68 from the second monomer of XPF homodimer (shown in circle).

vitro NER for damaged DNA, many of these acts through a dimeric complex, such as XPF-XPF homodimer, XPFERCC1 and archeal XPF homodimer. The available NMR structure of XPF homodimer presents a twofold symmetry. Due to the presence of several aromatic residues (Phe19, Phe68, Phe119, Phe168), the π-π interactions are formed on the interfaces of XPF-XPF homodimer which contribute toward the stability of this complex (Fig. 6) [100]. Meanwhile, β-amyloid peptide (Aβ) aggregation is the most acute pathological hallmark in Alzheimer’s disease [101]. The basic components of the amyloid are thin fibrils of a protein termed Aβ. Several studies have demonstrated that phenylalanine residues play a crucial role in the formation and selfassembly of amyloid fibril [102]. The stacking interactions between these residues are proven to be vital in Alzheimer’s disease [103]. Because of the importance of aromatic residues in fibril formation, researchers have attempted to block the self-assembly process of Aβ by breaking the stacking interactions between each subunit. The chemicals that can block π-stacking interactions may be the potential drugs for the control of Alzheimer’s disease [104]. Protein misfolding is one of the main reasons for neurodegenerative diseases, such as Alzheimer’s disease [105]. It is well established that protein folding is controlled by several inter- and intramolecular noncovalent interactions such as aromatic stacking. Aromatic residues stabilize local structure by stacking between near sequences while tertiary structure is formed by aromatic stacking between far sequences [106]. Research has been conducted to understand the atomic details of stacking interactions in misfolded proteins.

tion features/elements or structural elements (MoRFs/ MoREs) [109]. Recently, it has been documented that aromatic residues occur at lower frequencies in IDPs [110]. However, Mohan et al. suggests the presence of at least one aromatic amino acid in most MoRFs region [111]. A recent study of 77 protein-protein complexes of IDPs shows that 40% of the complexes contain at least two aromatic residues in stacking state [112]. These aromatic amino acids link binding and function of IDPs.

On the other hand, intrinsically disordered proteins (IDPs) are associated with many human diseases, including cancer, cardiovascular disease and neurodegenerative diseases [107]. IDPs lack well-defined structures of proteins and their conformations can fluctuate over time. Besides, they have the binding sites which can recognize multiple partners through adoption of different conformations [108]. These common binding sites are called molecular recogni-

ABBREVIATIONS

CONCLUSION The aromatic stacking interactions are common and diverse in nature. It is one of the main forces for the stabilization in the complexes of proteins and macromolecular ligands. In protein-protein complexes, the aromatic stacking can be found in the interfaces of different oligomers, such as dimer, trimer and tetramer. In protein-nucleotide complexes, the stacking interactions not only play a role in the recognition of binding site but also crucial in stabilizing the complex. Recently, in the emerging field of nanotechnology, the aromatic stacking has proven its value in the formation of protein-nanoparticle conjugate. Due to the importance of aromatic stacking, it is often involved in the pathogenesis of a variety of diseases, such as Alzheimer’s disease and cancer. Therefore, the study of aromatic stacking interactions in protein-ligand complexes will help researchers to understand the mechanisms behind many diseases and assist the design of new medicines.

Aβ

= β-amyloid peptide

CBC

= Cap-binding complex

Csp

= Cold shock proteins

Eps8

= Epidermal growth factor receptor pathway substrate 8

F (Phe)

= Phenylalanine

FPPs

= Farnesyl pyrophosphate synthase

Glu

= Glutamic acid

GSTs

= Glutathione transferases

H (His)

= Histidine

IDPs

= Intrinsically disordered proteins

MoRFs

= Molecular recognition features

mPGES-1 = Microsomal prostaglandin E synthase 1 NER

= Nucleotide excision repair

OPPs

= Octaprenyl pyrophosphate synthase

RRM

= RNA recognition motif

RRM

= RNA recognition motif (RRM)

SWCNT

= Single wall carbon nanotube

TZF

= Tandem zinc finger

W (Trp)

= Tryptophan

Y (Tyr)

= Tyrosine

[8]

[9] [10]

[11] [12]

[13]

[14]

CONFLICT OF INTEREST

[15]

The authors confirm that this article content has no conflict of interest.

[16]

ACKNOWLEDGEMENTS This work is financially supported by National Key Basic Research Program of China (No. 2013CB933900).

[17]

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