Aromatic stacking in the sugar binding site of the lactose permease

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.     

Effect of water coordination on competition between π and non-π cation binding sites in aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan Li+, Na+, and K+ complexes

Quantum chemistry methods have been applied to charged complexes of the alkali metals Li(+), Na(+), and K(+) with the aromatic amino acids (AAAs) phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). The geometries of 72 different complexes (Phe·M, Tyr·M, Trp·M, M is Li(+), Na(+), or K(+)) were completely optimized at the B3LYP/6-311+G(d,p) level of density functional theory. The solvent effect on the geometry and stability of individual complexes was studied by making use of a microsolvation model. The interaction enthalpies, entropies, and Gibbs energies of nine different complexes of the systems Phe·M, Tyr·M, and Trp·M (M is Li(+), Na(+), or K(+)) were also determined at the B3LYP density functional level of theory. The calculated Gibbs binding energies of the M(+)-AAA complexes follow the order Phe < Tyr < Trp for all three metal cations studied. Among the three AAAs studied, the indole ring of Trp is the best π donor for alkali metal cations. Our calculations demonstrated the existence of strong cation-π interactions between the alkali metals and the aromatic side chains of the three AAAs. These AAAs comprise about 8% of all known protein sequences. Thus, besides the potential for hydrogen-bond interaction, aromatic residues of Phe, Tyr, and Trp show great potential for π-donor interactions. The existence of cation-π interaction in proteins has also been demonstrated experimentally. However, more complex experimental studies of metal cation-π interaction in diverse biological systems will no doubt lead to more exact validation of these investigations.     

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of hydrophobic mismatch between phosphatidylcholine bilayers and transmembrane alpha-helical peptides depend on the nature of interfacially exposed aromatic and charged residues

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.     

Side-chain interactions in the C-peptide helix: Phe 8 ... His 12+

Previous studies have demonstrated that His 12 plays a major role in the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here, amino acid replacement experiments show that His 12+ stabilizes the C-peptide helix chiefly by interacting with Phe 8. The Phe 8 ... His 12+ ring interaction is specific for the protonated form of His 12 (His 12+) and the interaction is not screened significantly by NaCl, unlike the charged group ... helix dipole interactions studied earlier in C-peptide. Analogs of C-peptide that are unable to form the Phe 8 ... His 12+ interaction show large increases in helix content for Phe----Ala and His----Ala. Therefore, the helical tendencies of the individual residues Phe, His, and Ala are important in determining the result of a replacement experiment. Since the side chains of Phe 8 and His 12 probably interact within the N-terminal helix of ribonuclease A, the existence of the Phe 8 ... His 12+ interaction in the isolated C-peptide helix adds to the evidence that the C-peptide helix is an autonomous folding unit.     

Addition of side chain interactions to modified Lifson-Roig helix-coil theory: application to energetics of phenylalanine-methionine interactions.

We introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson-Roig helix-coil theory of Doig et al. (1994, Biochemistry 33:3396-3403). The helix/coil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields delta G for the Phe-Met orientation to be -0.75 kcal.mol-1, whereas that for the Met-Phe orientation is -0.54 kcal.mol-1. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe-Met i, i + 4 at the N-terminus, the C-terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain-side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix.     

Solution NMR structure of a model class A (apolipoprotein) amphipathic alpha helical peptide

To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH(2) (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d(3))/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C(beta)H protons (all the residues except Ala and Val) and gammaCH(3) (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic alpha helical structure of Ac-18A-NH(2) is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.     

Effect of the N2 residue on the stability of the α-helix for all 20 amino acids

N2 is the second position in the alpha-helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH(3)CO-[AXAAAAKAAAAKAAGY]-NH(2)) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 residue identity has been used to determine a free-energy scale by analysis with a modified Lifson-Roig helix-coil theory that includes a parameter for the N2 energy (n2). The rank order of DeltaDeltaG((relative to Ala)) is Glu(-), Asp(-) > Ala > Glu(0), Leu, Val, Gln, Thr, Ile, Ser, Met, Asp(0), His(0), Arg, Cys, Lys, Phe > Asn, > Gly, His(+), Pro, Tyr. The results correlate very well with N2 propensities in proteins, moderately well with N1 and helix interior preferences, and not at all with N-cap preferences. The strongest energetic effects result from interactions with the helix dipole, which favors negative charges at the helix N terminus. Hydrogen bonds to side chains at N2, such as Gln, Ser, and Thr, are weak, despite occurring frequently in protein crystal structures, in contrast to the N-cap position. This is because N-cap hydrogen bonds are close to linear, whereas N2 hydrogen bonds have poor geometry. These results can be used to modify protein stability rationally, help design helices, and improve prediction of helix location and stability.     

Investigations on C-H...pi interactions in RNA binding proteins

We have investigated the roles played by C-Hcdots, three dots, centeredpi interactions in RNA binding proteins. There was an average of 55 C-Hcdots, three dots, centeredpi interactions per protein and also there was an average of one significant C-Hcdots, three dots, centeredpi interaction for every nine residues in the 59 RNA binding proteins studied. Main-chain to side-chain C-Hcdots, three dots, centeredpi interactions is the predominant type of interactions in RNA binding proteins. The donor atom contribution to C-Hcdots, three dots, centeredpi interactions was mainly from Phe, Tyr, Trp, Pro, Gly, Lys, His and Ala residues. The acceptor atom contribution to main-chain to side-chain C-Hcdots, three dots, centeredpi and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions was mainly from Phe and Tyr residues. On the contrary, the acceptor atoms of Trp residues contributed to all the four types of C-Hcdots, three dots, centeredpi interactions. Also, Trp contributed both donor and acceptor atoms in main-chain to side-chain, main-chain to side-chain five-member aromatic ring and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions. The secondary structure preference analysis of C-Hcdots, three dots, centeredpi interacting residues showed that, Arg, Gln, Glu, His, Ile, Leu, Lys, Met, Phe and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Trp and Val preferred to be in strand conformation. Long-range C-Hcdots, three dots, centeredpi interactions are the predominant type of interactions in RNA binding proteins. More than 50% of C-Hcdots, three dots, centeredpi interacting residues had a higher conservation score. Significant percentage of C-Hcdots, three dots, centeredpi interacting residues had one or more stabilization centers. Seven percent of the theoretically predicted stabilizing residues were also involved in C-Hcdots, three dots, centeredpi interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

1H-NMR spectroscopic manifestations of ligand binding to the kringle 4 domain of human plasminogen

Structural aspects of the binding of the linear ligands N alpha-acetyl-L-lysine (AcLys) and epsilon-aminocaproic acid (epsilon ACA) and of the cyclic analogs trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) to the intact plasminogen kringle 4 domain have been investigated by 1H-NMR spectroscopy at 300 and 600 MHz. Ligand binding results in consistent shifts of the His-II (His31), Trp-I (Trp25?), Trp-II (Trp62?), Trp-III (Trp72), Tyr-II (Tyr50), and Phe64 ring signals. BASA tends to induce larger shifts than elicited by the aliphatic ligands, most noticeably on Trp-II and on Trp72, suggesting that the ligand aromatic ring interacts with the two indole groups. Trp-II and, to lesser extent, Trp-I interact with an acidic side chain group, in a manner that is blocked by BASA. BASA binding also perturbs Tyr-II (Tyr50), Tyr-III (Tyr41), and Tyr-IV (Tyr74) over a wide pH range and lowers the pKa* of His31 from approximately 4.8 to approximately 4.6. His-III (His33) responds to BASA and AMCHA but is relatively insensitive to the linear ligands. His33 carries a sterically shielded side chain which, in conjunction with Leu46, Trp-I, Tyr50, and Tyr74, participates in structuring the kringle hydrophobic core, contiguous to the binding site. Pronounced shifts are observed for aliphatic resonances stemming from the kringle-bound molecules of AMCHA, AcLys, and epsilon ACA. It is proposed that the lysine-binding site is mostly supported by the loop that extends from Cys51 through Cys71 and that aromatic residues, which include Trp-II, Trp72, and Phe64, play a major role in interacting with the nonpolar segment of the ligand molecule. The binding site also encompasses Tyr50, Tyr74, His31, and His33 although it is not clear the extent to which these residues interact directly with the ligand.     

NMR study of the structural characteristics of variants of yeast iso-1-cytochrome c in which unvaried aromatic residues have been substituted.

The structures of variants of yeast iso-1-cytochrome c, in which the previously unchanged Tyr48 and Tyr48 + Trp59 have been replaced by Phe, have been characterised by NMR. The NMR data indicated that the structures of the variant cytochromes c are very similar to the wild-type protein. In particular, the heme environment and interactions of the heme macrocycle were shown to be preserved. The observation of chemical shift differences have allowed for the assessment of conformational changes. The substitution of Trp59 by Phe may have caused a small conformational change, a manifestation of which is the observed chemical shift differences at His39, Val57 and Tyr74. The structural basis for the reduction in redox potential accompanying the amino acid substitutions is discussed and the proposal made that the changes in potential are a direct consequence of the side chain properties and do not result primarily from conformational changes.     

Arginine and Lysine interactions with �� residues in metalloproteins

Metalloproteins have many different functions in cells such as enzymes; signal transduction, transport and storage proteins. About one third of all proteins require metals to carry out their functions. In the present study we have analyzed the roles played by Arg and Lys (cationic side chains) interactions with π (Phe, Tyr or Trp) residues and their role in the structural stability of metalloproteins. These interactions might play an important role in the global conformational stability in metalloproteins. In spite of its lower natural occurrence (1.76%) the number of Trp residues involved in energetically significant interactions is higher in metalloproteins.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

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.     

Importance of the conserved aromatic residues in the scorpion alpha-like toxin BmK M1: the hydrophobic surface region revisited

About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded beta-sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the beta2-sheet and in the loop between the beta2- and beta3-sheets, respectively) are most likely involved in the pharmacological function of the toxin.     

Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.     

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.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.     

The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution

An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus. The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal. The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169. Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167. The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32. Gln 150 forms a bridge between Tyr 36 and Trp 131. These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases. Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III). These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28. The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix. The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion. Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit. Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156. The center of the oligomeric molecule is filled with solvent.     

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.