Non-hydrogen bond interactions involving the methionine sulfur atom

Of all the nonbonded interactions, hydrogen bond, because of its geometry involving polar atoms, is the most easily recognizable. Here we characterize two interactions involving the divalent sulfur of methionine (Met) residues that do not need any participation of proton. In one an oxygen atom of the main-chain carbonyl group or a carboxylate side chain is used. In another an aromatic atom interacting along the face of the ring is utilized. In these, the divalent sulfur behaves as an electrophile and the other electron-rich atom, a nucleophile. The stereochemistry of the interaction is such that the nucleophile tends to approach approximately along the extension of one of the covalent bonds to S. The nitrogen atom of histidine side chain is extensively used in these nonbonded contacts. There is no particular geometric pattern in the interaction of S with the edge of an aromatic ring, except when an N-H group in involved, which is found within 40 degrees from the perpendicular to the sulfide plane, thus defining the geometry of hydrogen bond interaction involving the sulfur atom. As most of the Met residues which partake in such stereospecific interactions are buried, these would be important for the stability of the protein core, and their incorporation in the binding site would be useful for molecular recognition and optimization of the site's affinity for partners (especially containing aromatic and heteroaromatic groups). Mutational studies aimed at replacing Met by other residues would benefit from the delineation of these interactions.     

A theoretical and experimental case study of the hydrogen bonding predilection of S-methylcysteine

S-containing amino acids can lead to two types of local NH···S interactions which bridge backbone NH sites to the side chain to form either intra- or inter-residue H-bonds. The present work reports on the conformational preferences of S-methyl-l-cysteine, Cys(Me), using a variety of investigating tools, ranging from quantum chemistry simulations, gas-phase UV and IR laser spectroscopy, and solution state IR and NMR spectroscopies, on model compounds comprising one or two Cys(Me) residues. We demonstrate that in gas phase and in low polarity solution, the C- and N-capped model compound for one Cys(Me) residue adopts a preferred C5–C6γ conformation which combines an intra-residue N–H···O=C backbone interaction (C5) and an inter-residue N–H···S interaction implicating the side-chain sulfur atom (C6γ). In contrast, the dominant conformation of the C- and N-capped model compound featuring two consecutive Cys(Me) residues is a regular type I β-turn. This structure is incompatible with concomitant C6γ interactions, which are no longer in evidence. Instead, C5γ interactions occur, that are fully consistent with the turn geometry and additionally stabilize the structure. Comparison with the thietane amino acid Attc, which exhibits a rigid cyclic side chain, pinpoints the significance of side chain flexibility for the specific conformational behavior of Cys(Me).

     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Hypothesis of a proton switch in QM/MM modelling of interaction of dUMP analogues with thymidylate synthase

Quantum mechanical, molecular mechanics and molecular dynamics (MD) methods were used to investigate initial steps of 2′-deoxyuridine-5′-monophosphate (dUMP) methylation catalysed by the thymidylate synthase (TS) enzyme. The amino acid residues surrounding the active site within a 10 Å radius sphere were modelled with the combined quantum mechanical (B3LYP/LANL2DZ) and molecular mechanics ONIOM double-layer method. The results indicated the initial nucleophilic attack of Cys146 on dUMP to be concerted with formation of a hydrogen bond to the oxygen O4 of dUMP. Moreover, the proton in the vicinity of the O4 atom appears to act as a ‘proton switch’: if a proton is present near O4, it stabilises the S(Cys146)–C6(dUMP) sulphur–carbon bond, but if it is absent, the sulphur–carbon bond does not form. If the O4 oxygen is replaced by sulphur atom, the ‘switch effect’ does not occur. The suggested correlation between the strength of hydrogen bond involving O4 oxygen and the ability of dUMP to form bonds at C6 corresponds well to the crystal structures of TS complexes available in the Protein Data Bank. In the vast majority of crystal structures, the presence of the S(Cys146)–C6(dUMP) bond was coupled with the presence of hydrogen bond between the dUMP O4 atom and the conserved Asn177. The ‘proton switch’ hypothesis is supported also by the results of MD studies of TS binary complexes, suggesting that average distance separating S(Cys146) and C6(dUMP) becomes distinctly shorter in the presence of hydrogen bonding between Asn177 and O4.     

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.     

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.     

Refined 2.5 Å X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor: Crystallization,patterson search,structure determination,refinement, structure and comparison with its components and with the bovine trypsin-pancreatic trypsin inhibitor complex

The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P4₁2₁2 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al., 1974) and the structure of porcine pancreatic kallikrein A (Bode et al., 1983). The kallikrein-PTI model has been crystallographically refined to an R-value of 0·23 including X-ray data to 2·5 Å.The root-mean-square deviation, including all main-chain atoms, is 0·45 Å and 0·65 Å for the PTI and for the kallikrein component, respectively, compared with the refined models of the free components. The largest differences are observed in external loops of the kallikrein molecule surrounding the binding site, particularly in the C-terminal part of the intermediate helix around His172. Overall, PTI binding to kallikrein is similar to that of the trypsin complex. In particular, the conformation of the groups at the active site is identical within experimental error (in spite of the different pH values of the two structures). Ser195 OG is about 2·5 Å away from the susceptible inhibitor bond Lys15 C and forms an optimal 2·5 Å hydrogen bond with His57 NE.The PTI residues Thr11 to Ile18 and Val34 to Arg39 are in direct contact with kallikrein residues and form nine intermolecular hydrogen bonds. The reactive site Lys15 protrudes into the specificity pocket of kallikrein as in the trypsin complex, but its distal ammonium group is positioned differently to accommodate the side-chain of Ser226. Ser226 OG mediates the ionic interaction between the ammonium group and the carboxylate group of Asp189. Model-building studies indicate that an arginine side-chain could be accommodated in this pocket. The PTI disulfide bridge 14–38 forces the kallikrein residue Tyr99 to swing out of its normal position. Model-building experiments show that large hydrophobic residues such as phenylalanine can be accommodated at this (S2) site in a wedge-shaped hydrophobic cavity, which is formed by the indole ring of Trp215 and by the phenolic side-chain of Tyr99, and which opens towards the bound inhibitor/substrate chain. Arg17 in PTI forms a favorable hydrogen bond and van der Waals' contacts with kallikrein residues, whereas the additional hydrogen bond formed in the trypsin-PTI complex between Tvr39 OEH and Ile19 N is not possible The kallikrein binding site offers a qualitative explanation of the unusual binding and cleavage at the N-terminal Met-Lys site of kininogen. Model-building experiments suggest that the generally restricted capacity of kallikrein to bind protein inhibitors with more extended binding segments might be explained by steric hindrance with some extruding external loops surrounding the kallikrein binding site (Bode et al., 1983).     

The occurrence of C--H...O hydrogen bonds in alpha-helices and helix termini in globular proteins

A comprehensive database analysis of C--H...O hydrogen bonds in 3124 alpha-helices and their corresponding helix termini has been carried out from a nonredundant data set of high-resolution globular protein structures resolved at better than 2.0 A in order to investigate their role in the helix, the important protein secondary structural element. The possible occurrence of 5 --> 1 C--H...O hydrogen bond between the ith residue CH group and (i - 4)th residue C==O with C...O < or = 3.8 A is studied, considering as potential donors the main-chain Calpha and the side-chain carbon atoms Cbeta, Cgamma, Cdelta and Cepsilon. Similar analysis has been carried out for 4 --> 1 C--H...O hydrogen bonds, since the C--H...O hydrogen bonds found in helices are predominantly of type 5 --> 1 or 4 --> 1. A total of 17,367 (9310 of type 5 --> 1 and 8057 of type 4 --> 1) C--H...O hydrogen bonds are found to satisfy the selected criteria. The average stereochemical parameters for the data set suggest that the observed C--H...O hydrogen bonds are attractive interactions. Our analysis reveals that the Cgamma and Cbeta hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic beta-branched residue Ile to participate in 5 --> 1 C--H...O hydrogen bonds involving methylene Cgamma 1 atom as donor in alpha-helices. This may be an enthalpic compensation for the greater loss of side-chain conformational entropy for beta-branched amino acids due to the constraint on side-chain torsion angle, namely, chi1, when they occur in helices. The preference of amino acids for 4 --> 1 C--H...O hydrogen bonds is found to be more for Asp, Cys, and for aromatic residues Trp, Phe, and His. Interestingly, overall propensity for C--H...O hydrogen bonds shows that a majority of the helix favoring residues such as Met, Glu, Arg, Lys, Leu, and Gln, which also have large side-chains, prefer to be involved in such types of weak attractive interactions in helices. The amino acid side-chains that participate in C--H...O interactions are found to shield the acceptor carbonyl oxygen atom from the solvent. In addition, C--H...O hydrogen bonds are present along with helix stabilizing salt bridges. A novel helix terminating interaction motif, X-Gly with Gly at C(cap) position having 5 --> 1 Calpha--H...O, and a chain reversal structural motif having 1 --> 5 Calpha-H...O have been identified and discussed. Our analysis highlights that a multitude of local C--H...O hydrogen bonds formed by a variety of amino acid side-chains and Calpha hydrogen atoms occur in helices and more so at the helix termini. It may be surmised that the main-chain Calpha and the side-chain CH that participate in C--H...O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N--H...O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.     

Proton polarizability and proton transfer in histidine-phosphate hydrogen bonds as a function of cations present: ir investigations

(L -His)_n- dihydrogen phosphate systems are studied by ir spectroscopy in the presence of various cations and as a function of the degree of hydration. Ir continua indicate that (I) OH … N ? O^?…H⁺N (IIR) hydrogen bonds are formed and that these bonds show high proton polarizability, which increases from the Li⁺ to the K⁺ system. In the K⁺?system, His-P_i-P_i chains are formed, showing particularly high proton polarizability due to collective proton motion within both hydrogen bonds. The OH N ? O^?…H^?N equilibria are determined from ir bands. With the Li⁺ system, 55% of the protons are present at the histidine residues; this percentage is smaller with the Na⁺ system (41%), and amounts to only 32% with the K⁺ system. With the increasing degree of hydration the removal of the degeneracy of ν_as?PO^2?₃ vanishes, indicating loosening of the cations from the phosphates. Nevertheless, the hydrogen bond acceptor O atom becomes more negative; a shift of the equilibrium to the right is observed in the OH… N ? O^?…H⁺N bond. This is explained by the strong interaction of the dipole of the hydrogen bonds with the water molecules. All these results show that protons can be shifted easily in these hydrogen bonds due to their high proton polarizability. The transfer equilibria can be controlled easily by local electrical fields. In addition, these results may be of significance when phosphates interact with proteins.     

An electrophile-nucleophile interaction in metalloprotein structures.

A new attractive interaction in metalloprotein structures, between the thiolate anion of a metal-bound cysteine (acting as a nucleophile) and a carbonyl carbon of a peptide group (an electrophile), has been identified. From 82 cases extracted from 23 metalloprotein structures, the interacting S and C atoms are found to be at a distance of 3.2 (+/-2) A, such that the angle S ... C-O is 109 degrees (+/-15 degrees). Usually, the interacting atoms are from the same Cys residue, and to allow the S to interact with the carbonyl group the side-chain and the main-chain torsion angles deviate from those found in cysteines not bound by metals. There is a good correlation between the S...C distance and the angular deviation of the S...C vector from the normal to the peptide plane. Various data points may be envisaged to represent "snapshots" along the reaction coordinate for the intra-residue attack of Cys S on the CO group.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

A theoretical investigation on the conformation and the interaction of CHF2OCF2CHF2 (desflurane II) with one water molecule

The conformation and the interaction of CHF₂OCF₂CHF₂ (desflurane II) with one water molecule is investigated theoretically using the ab initio MP2/aug-cc-pvdz and DFT-based M062X/6-311++G(d,p) methods. The calculations include the optimized geometries, the harmonic frequencies of relevant vibrational modes along with a natural bond orbital (NBO) analysis including the NBO charges, the hybridization of the C atom and the intra- and intermolecular hyperconjugation energies. In the two most stable conformers, the CH bond of the F₂HCO- group occupies the gauche position. The hyperconjugation energies are about the same for both conformers and the conformational preference depends on the interaction between the non-bonded F and H atoms. The deprotonation enthalpies of the CH bonds are about the same for both conformers, the proton affinity of the less stable conformer being 3 kcal mol^?1 higher. Both conformers of desflurane II interact with water forming cyclic complexes characterized by CH…O and OH…F hydrogen bonds. The binding energies are moderate, ranging from ?2.4 to ?3.2 kcal mol^?1 at the MP2 level. The origin of the blue shifts of the ν(CH) vibrations is analyzed. In three of the complexes, the water molecule acts as an electron donor. Interestingly, in these cases a charge transfer is also directed to the non bonded OH group of the water molecule. This effect seems to be a property of polyfluorinated ethers.     

1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

6-Azauridine, a nucleoside with unusual ribose conformation. The molecular and crystal structure

The cytostatic analogue ribo-6-azauridine crystallizes in the orthorhombic space group P2₁2₁2₁ with eight molecules per unit cell of dimensions $\text{a = 20.230, b = 7.709, c = 12.863}\text{A}\text{?}$. A trial structure was obtained by direct methods. Least-squares refinement of co-ordinates and anisotropic thermal parameters based on 1998 reflections measured on a four-circle diffractometer led to a discrepancy index R = 4.0%. Like uridine, 6-azauridine has the anti conformation about the glycosidic bond and a C(3′)-endo sugar pucker. Unlike uridine, it exhibits a close approach of N(6) to C(2′) at only 2.814 and 2.844 Å in the two independent molecules, and a C(5′)(5′) bond that is gauche to C(4′)O(1′) but trans to C(4′)C(3′); this conformation about a C(4′)C(5′) bond has never been observed before for C(3′)-endo puckered riboses in the crystalline state. The crystal structure displays a pseudo-A face centering and very similar conformational parameters for the two independent molecules. Every OH and NH group in the structure serves as a proton donor in a hydrogen bond, including an unusual N(3)—H(3) … O(1′) link. Molecular orbital calculations by the extended Hückel method indicate that from uridine to 6-azauridine the net charge changes sign at ring positions 5 and 6 and disappears at 1.     

Application of CNDO2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

$\text{CNDO}\text{2}$ molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond distance = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond distance = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

A study of dihydrogen bond interactions through three-centre bond and group indices

We report here an investigation into the correlation between dihydrogen bond energies, three-centre bond indices and group indices in some dihydrogen-bonded dimers. This kind of bond is generated by interaction between proton-donator and proton-acceptor groups, XH^σ+…H′^σ ? M, where X is a more electronegative atom and M a less electronegative atom than hydrogen. The different electronegativities of the X atoms, as well the M atoms, would affect the correlations between H^σ+…H′^σ ?  distances and bond energies of these systems. In this work it will be shown that three-centre bond indices and group indices exhibit a better correlation with bond energies when compared to H^σ+…H′^σ ?  distances for this kind of system.     

Characterization of beta-turn and Asx-turns mimicry in a model peptide: stabilization via C--H . . . O interaction

The chemical synthesis and single-crystal X-ray diffraction analysis of a model peptide, Boc-Thr-Thr-NH2 (1) comprised of proteinogenic residues bearing an amphiphilic Cbeta -stereogenic center, has been described. Interestingly, the analysis of its molecular structure revealed the existence of a distinct conformation that mimics a typical beta-turn and Asx-turns, i.e., the two Thr residues occupy the left- and right-corner positions. The main-chain torsion angles of the N- and C-terminal residues i.e., semiextended: phi = -68.9 degrees , psi = 128.6 degrees ; semifolded: phi = -138.1 degrees , psi = 2.5 degrees conformations, respectively, in conjunction with a gauche- disposition of the obligatory C-terminus Thr CgammaH3 group, characterize the occurrence of the newly described beta-turn- and Asx-turns-like topology. The preferred molecular structure is suggested to be stabilized by an effective nonconventional main-chain to side-chain Ci=O . . . H--Cgamma(i+2)-type intraturn hydrogen bond. Noteworthy, the observed topology of the resulting 10-membered hydrogen-bonded ring is essentially similar to the one perceived for a classical beta-turn and the Asx-turns, stabilized by a conventional intraturn hydrogen bond. Considering the signs as well as magnitudes of the backbone torsion angles and the orientation of the central peptide bond, the overall mimicked topology resembles the type II beta-turn or type II Asx-turns. An analysis of Xaa-Thr sequences in high-resolution X-ray elucidated protein structures revealed the novel topology prevalence in functional proteins (unpublished). In view of indubitable structural as well as functional importance of nonconventional interactions in bioorganic and biomacromolecules, we intend to highlight the participation of Thr CgammaH in the creation of a short-range C=O . . . H--Cgamma -type interaction in peptides and proteins.     

Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida

Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.