Role of a two-residue spacer in an alpha,beta-Didehydrophenylalanine containing hexapeptide: crystal and solution structure of Boc-val-deltaPhe-Leu-Ala-deltaPhe-Ala-OMe.

The peptide Boc-Val1-deltaPhe2-Leu3-Ala4-deltaPhe5-Ala6-OMe has been examined for the structural consequence of placing a two-residue segment between the deltaPhe residues. The peptide is stabilized by four consecutive beta-turns. The overall conformation of the molecule is a right-handed 3(10)-helix, with average (phi, psi) values (-67.7 degrees, -22.7 degrees), unwound at the C-terminus. The 1H NMR results also suggest that the peptide maintains its 3(10)-helical structure in solution as observed in the crystal state. The crystal structure is stabilized through head-to-tail hydrogen bonds and a repertoire of aromatic interactions laterally directed between adjacent helices, which are antiparallel to each other. The aromatic ring of deltaPhe5 forms the hub of multicentred interactions, namely as a donor in aromatic C-H...pi and aromatic C-H...O=C interactions and as an acceptor in a CH3...pi interaction. The present structure uniquely illustrates the unusual capability of a deltaPhe ring to host such concerted interactions and suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures.     

Geometry of nonbonded interactions involving planar groups in proteins

Although hydrophobic interaction is the main contributing factor to the stability of the protein fold, the specificity of the folding process depends on many directional interactions. An analysis has been carried out on the geometry of interaction between planar moieties of ten side chains (Phe, Tyr, Trp, His, Arg, Pro, Asp, Glu, Asn and Gln), the aromatic residues and the sulfide planes (of Met and cystine), and the aromatic residues and the peptide planes within the protein tertiary structures available in the Protein Data Bank. The occurrence of hydrogen bonds and other nonconventional interactions such as C-H...pi, C-H...O, electrophile-nucleophile interactions involving the planar moieties has been elucidated. The specific nature of the interactions constraints many of the residue pairs to occur with a fixed sequence difference, maintaining a sequential order, when located in secondary structural elements, such as alpha-helices and beta-turns. The importance of many of these interactions (for example, aromatic residues interacting with Pro or cystine sulfur atom) is revealed by the higher degree of conservation observed for them in protein structures and binding regions. The planar residues are well represented in the active sites, and the geometry of their interactions does not deviate from the general distribution. The geometrical relationship between interacting residues provides valuable insights into the process of protein folding and would be useful for the design of protein molecules and modulation of their binding properties.     

C-H...pi-interactions in proteins

A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring.The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed.It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.     

Aromatic-aromatic interactions in and around alpha-helices

To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in alpha-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H...pi and N-H...pi) which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An alpha-helix and a contiguous 3(10)-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side.     

Hydrogen bonds with pi-acceptors in proteins: frequencies and role in stabilizing local 3D structures

A comprehensive structural analysis of X--H...pi hydrogen bonding in proteins is performed based on 592 published high-resolution crystal structures (< or = 1.6 A). All potential donors and acceptors are considered, including acidic C--H groups. The sample contains 1311 putative X--H...pi hydrogen bonds with N--H, O--H or S--H donors, that is about one per 10.8 aromatic residues. By far the most efficient pi-acceptor is the side-chain of Trp, which accepts one X--H...pi hydrogen bond per 5.7 residues. The focus of the analysis is on recurrent structural patterns involving regular secondary structure elements. Numerous examples are found where peptide X--H...pi interactions are functional in stabilization of helix termini, strand ends, strand edges, beta-bulges and regular turns. Side-chain X--H...pi hydrogen bonds are formed in considerable numbers in alpha-helices and beta-sheets. Geometrical data on various types of X--H...pi hydrogen bonds are given.     

Enhanced preference for pi-bond containing substrates is correlated to Pro110 in the substrate-binding tunnel of Escherichia coli thioesterase I/protease I/lysophospholipase L(1)

Escherichia coli thioesterase I/protease I/lysophospholipase L(1) (TAP) possesses multifunctional enzyme with thioesterase, esterase, arylesterase, protease, and lysophospholipase activities. Leu109, located at the substrate-binding tunnel, when substituted with proline (Pro) in TAP, shifted the substrate-preference from medium-to-long acyl chains to shorter acyl chains of triglyceride and p-nitrophenyl ester, and increased the preference for aromatic-amino acid-derived esters. In the three-dimensional TAP structures, the only noticeable alteration of backbone and side chain conformation was located at the downstream Pro110-Ala123 region rather than at Pro109 itself. The residue Pro110, adjacent to Leu109 or Pro109, was found to contribute to the substrate preference of TAP enzymes for esters containing acyl groups with pi bond(s) or aromatic group(s). Some of the interactions between the enzyme protein and the substrate may be contributed by an attractive force between the Pro110 C-H donor and the substrate pi-acceptor.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.     

Insights into the role of the aromatic residue in galactose-binding sites: MP2/6-311G++** study on galactose- and glucose-aromatic residue analogue complexes

The presence of an aromatic residue (Trp, Phe, Tyr) facing the nonpolar face of galactose is a common feature of galactose-specific lectins. The interactions such as those between the C-H groups of galactose and the pi-electron cloud of aromatic residues have been characterized as weak hydrogen bonds between soft acids and soft bases, largely governed by dispersive and charge transfer interactions. An analysis of the binding sites of several galactose-specific lectins revealed that the spatial position-orientation of galactose relative to the binding site aromatic residue varies substantially. The effect of variations in position-orientations of galactose on the interaction energies of galactose-aromatic residue complexes has not been determined so far. In view of this, MP2/6-311G++** calculations were performed on galactose- and glucose-aromatic residue analogue complexes in eight position-orientations. The results show that the strength of the C-H...pi interactions in galactose-aromatic residue complexes is comparable to that of a hydrogen bond. Rather than the type of aromatic residue, the position-orientation of the saccharide appears to be more critical in determining the strength of their interactions. Earlier studies have found the binding site aromatic residue to be critical, but its role was not clear. This study shows that the aromatic residue is important for discriminating galactose from glucose, in addition to its contribution to binding energy.     

A neutral molecule in a cation-binding site: specific binding of a PEG-SH to acetylcholinesterase from Torpedo californica

The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.     

Tryptophan-containing peptide helices: interactions involving the indole side chain.

Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe, 1) as well as i and i + 6 (Boc-Leu-Trp-Val-Aib-Ala-Aib-Leu-Trp-Val-OMe, 2) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen-bonded NH groups in peptides 1 and 2, respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X-ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain-backbone hydrogen bonding involving the indole Nepsilon1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge-to-face aromatic interactions between the indoles suggest a potential C-H...pi interaction involving the Czeta3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) Nepsilon1H protons, presumably in a manner similar to that observed in the crystal.     

The importance of CH/pi interactions to the function of carbohydrate binding proteins

It is suggested that the interactions between the hydrophobic C-H groups of carbohydrate residues and the pi-electron systems of aromatic amino-acid residues play an important role in the ligand-recognition function of carbohydrate-binding proteins. This review focuses on our recent structural and functional studies of human lysozyme and hevein-domain type lectins (wheat-germ agglutinin and Ac-AMP2) aimed at understanding how CH/pi interactions are involved in the actual binding events.     

Non-classical hydrogen bonds in interleukins: the role of C-H...O interactions

The role of classical hydrogen bonds in the structural stability of biological macro-molecules is well understood. In the present study, we explore the influence of C-H...O interactions in relation to other environmental preferences in interleukins. Main chain-main chain interactions are predominant. Pro residues might stabilize helices and strands by C-H...O H-bonds in interleukins. Majority of the C-H...O interacting residues were solvent exposed. 62% of C-H...O interactions was long-range interactions. The results presented in this study might be useful for structural stability studies in interleukins.     

Geometry of nonbonded interactions involving planar groups in proteins

Although hydrophobic interaction is the main contributing factor to the stability of the protein fold, the specificity of the folding process depends on many directional interactions. An analysis has been carried out on the geometry of interaction between planar moieties of ten side chains (Phe, Tyr, Trp, His, Arg, Pro, Asp, Glu, Asn and Gln), the aromatic residues and the sulfide planes (of Met and cystine), and the aromatic residues and the peptide planes within the protein tertiary structures available in the Protein Data Bank. The occurrence of hydrogen bonds and other nonconventional interactions such as C–H⋯π, C–H⋯O, electrophile–nucleophile interactions involving the planar moieties has been elucidated. The specific nature of the interactions constraints many of the residue pairs to occur with a fixed sequence difference, maintaining a sequential order, when located in secondary structural elements, such as α-helices and β-turns. The importance of many of these interactions (for example, aromatic residues interacting with Pro or cystine sulfur atom) is revealed by the higher degree of conservation observed for them in protein structures and binding regions. The planar residues are well represented in the active sites, and the geometry of their interactions does not deviate from the general distribution. The geometrical relationship between interacting residues provides valuable insights into the process of protein folding and would be useful for the design of protein molecules and modulation of their binding properties.     

Contributions of cation-π interactions to the collagen triple helix stability

Cation–π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X–Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation–π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host–guest peptide system: (Pro-Hyp-Gly)₃-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)₃, where X is an aromatic residue and Y is a cationic residue, to study the cation–π interaction in the collagen triple helix. Circular dichroism (CD) measurements and T_m data analysis show that the cation–π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation–π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation–π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π–electron density and the cation–π interaction in collagen. Our data demonstrate that the cation–π interaction could play an important role in stabilizing the collagen triple helix.     

Computation of non-covalent interactions in TNF proteins and interleukins

The roles played by the non-covalent interactions have been investigated for a set of six TNF proteins and nine Interleukins. The stabilizing residues have been identified by a consensus approach using the concepts of available surface area, medium and long-range interactions and conservation of amino acid residues. The cation-pi interactions have been computed based on a geometric approach such as distance and energy criteria. We identified an average of 1 energetically significant cation-pi interactions in every 94 residues in TNF proteins and 1 in every 62 residues in Interleukins. In TNF proteins, the cationic groups Lys preferred to be in helix while Arg preferred to be in strand regions while in Interleukins the Arg residues preferred to be in helix and Lys preferred to be in strand regions. From the available surface area calculations, we found that, almost all the cation and pi residues in TNF proteins and Interleukins were either in buried or partially buried regions and none of them in the exposed regions. Medium and long-range interactions were predominant in both TNF proteins and Interleukins. It was observed that the percentage of stabilizing centers were more in TNF proteins as compared to the Interleukins, while the percentage of conserved residues were more in Interleukins than in TNF proteins. In the stabilizing residues Lys was observed to be a stabilizing residue in both TNF proteins and Interleukins. Among the aromatic group, Phe was seen to be a stabilizing residue in both TNF and Interleukins. We suggest that this study on the computation of cation-pi interactions in TNF proteins and Interleukins would be very helpful in further understanding the structure, stability and functional similarity of these proteins.     

A C-H triplebond O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H cdots, three dots, centered O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H triplebond O interaction between the T-4 C(alpha)H and T+1 Cz doublebond O group (C triplebond O< or =3.5A) becomes possible only when the T+1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T-4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T-4 position with the motif being further stabilized by the formation of a side-chain-backbone O triplebond H-N hydrogen bond involving the side-chain of residue T-4 and the N-H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H triplebond O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H cdots, three dots, centered O hydrogen bonds between (T-4) C(alpha)H triplebond O (T+1) and (T-8) C(alpha)H triplebondC doublebond O (T+3).     

Interstrand pairing patterns in beta-barrel membrane proteins: the positive-outside rule, aromatic rescue, and strand registration prediction

beta-Barrel membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Little is known about how residues in membrane beta-barrels interact preferentially with other residues on adjacent strands. We have developed probabilistic models to quantify propensities of residues for different spatial locations and for interstrand pairwise contact interactions involving strong H-bonds, side-chain interactions, and weak H-bonds. Using the reference state of exhaustive permutation of residues within the same beta-strand, the propensity values and p-values measuring statistical significance are calculated exactly by analytical formulae we have developed. Our findings show that there are characteristic preferences of residues for different membrane locations. Contrary to the "positive-inside" rule for helical membrane proteins, beta-barrel membrane proteins follow a significant albeit weaker "positive-outside" rule, in that the basic residues Arg and Lys are disproportionately favored in the extracellular cap region and disfavored in the periplasmic cap region. We find that different residue pairs prefer strong backbone H-bonded interstrand pairings (e.g. Gly-aromatic) or non-H-bonded pairings (e.g. aromatic-aromatic). In addition, we find that Tyr and Phe participate in aromatic rescue by shielding Gly from polar environments. We also show that these propensities can be used to predict the registration of strand pairs, an important task for the structure prediction of beta-barrel membrane proteins. Our accuracy of 44% is considerably better than random (7%). It also significantly outperforms a comparable registration prediction for soluble beta-sheets under similar conditions. Our results imply several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of beta-barrel membrane proteins. The propensity scales developed in this study will also be useful for computational structure prediction and for folding simulations.     

Role of CH/pi interactions in substrate binding by Escherichia coli beta-galactosidase

Interactions between carbohydrates and aromatic amino-acid residues are often observed in structures of carbohydrate-protein complexes. They are characterized by an orientation of the pyranose or furanose ring parallel with the aromatic ring of amino-acid residues. An important role in the formation of these complexes is supposed to be played by CH/pi interactions. This paper presents an ab initio quantum chemistry study of CH/pi interactions between beta-galactosidase from E. coli and its substrates and products. The energy stabilizing the interaction between Trp999 residue and substrate bound in the shallow binding mode was calculated at the MP2/6-31+G(d) level as 5.2kcalmol(-1) for the glucose moiety of allolactose, 2.4kcalmol(-1) for the galactose moiety of allolactose and 5.0kcalmol(-1) for the glucose moiety of lactose. The energy stabilizing the interaction between Trp568 residue and galactose in the deep binding mode was calculated as 2.7kcalmol(-1). Interaction energies at the HF/6-31+G(d) and B3LYP/6-31+G(d) levels were small or repulsive; therefore, highly correlated ab initio methods were necessary to study these interactions. These unexpectedly strong interactions give a rationale for allolactose formation and illustrate the role of the Trp999 residue. In addition, this illustrates the importance of CH/pi interactions for the function of carbohydrate-binding proteins and carbohydrate-processing enzymes.     

Conformation of peptides constructed from achiral amino acid residues Aib and DeltaZPhe: computational study of the effect of L/D- Leu at terminal positions

Conformational studies of the peptides constructed from achiral amino acid residues Aib and Delta(Z)Phe (I) Ac-Aib-Delta(Z)Phe-NHMe (II), and Ac-(Aib-Delta(Z)Phe)(3)-NHMe; peptides III-VI having L-Leu or D-Leu at either the N- or the C-terminal position and of peptides VII-X having Leu residues in different enantiomeric combinations at both the N- and the C-terminal positions in peptide II have been studied to design the peptide with the required helical sense. Peptide II, as expected, adopts degenerate left- and right-handed helical structures. It has been shown that the peptides IV and VI having D-Leu at either the N or the C terminus can be realized in the right-handed helical structure with the phi,psi values of -20 degrees and -60 degrees for the Aib/Delta(Z)Phe residues. L-Leu and D- Leu at both the terminals in peptides VII and VIII, respectively, have hardly any effect as both the left- and the right-handed structures are found to be degenerate. Peptides III and IX can be realized in right- and left-handed helical structures, respectively, in solvents of low polarity whereas peptides V and X are predicted to be in the right-handed helical structures stabilized by carbonyl-carbonyl interactions without the formation of hydrogen bonds. The conformational states with the phi,psi values of 0 degrees and -85 degrees in peptide V are characterized by rise per residue of 2.03 A, rotation per residue of 117.5 degrees , and 3.06 residues per turn. In all peptides having Leu residue at the N terminus, the methyl moiety of the acetyl group is involved in the CH/pi interactions with the Cepsilon--Cdelta edge of the aromatic ring of Delta(Z)Phe (3) and the amino group NH of Delta(Z)Phe is involved in the NH/pi interactions with its own aromatic ring. The CH(3) groups of the Aib residues are also involved in CH/pi interactions with the i + 1th and i + 3th Delta(Z)Phe's aromatic side chains.     

Registering alpha-helices and beta-strands using backbone C-H...O interactions

The possible occurrence of a novel helix terminating structural motif in proteins involving a stabilizing short C-H...O interaction has been examined using a dataset of 634 non-homologous protein structures (相似文献