Analogs of S-Adenosyl-L-Methionine in Studies of Methyltransferases.

Methyltransferases (MTases) play an important role in the functioning of living systems, catalyzing the methylation reactions of DNA, RNA, proteins, and small molecules, including endogenous compounds and drugs. Many human diseases are associated with disturbances in the functioning of these enzymes; therefore, the study of MTases is an urgent and important task. Most MTases use the cofactor S­adenosyl­L­methionine (SAM) as a methyl group donor. SAM analogs are widely applicable in the study of MTases: they are used in studies of the catalytic activity of these enzymes, in identification of substrates of new MTases, and for modification of the substrates or substrate linking to MTases. In this review, new synthetic analogs of SAM and the problems that can be solved with their usage are discussed.

[Analogs of S-Adenosyl-L-Methionine in Studies of Methyltransferases].

Methyltransferases (MTases) play an important role in the functioning of living systems, catalyzing the methylation reactions of DNA, RNA, proteins, and small molecules, including endogenous compounds and drugs. Many human diseases are associated with disturbances in the functioning of these enzymes; therefore, the study of MTases is an urgent and important task. Most MTases use the cofactor S-adenosyl-L-methionine (SAM) as a methyl group donor. SAM analogs are widely applicable in the study of MTases: they are used in studies of the catalytic activity of these enzymes, in identification of substrates of new MTases, and for modification of the substrates or substrate linking to MTases. In this review, new synthetic analogs of SAM and the problems that can be solved with their usage are discussed.

[Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms].

Intact amyloid-ß peptides (Aß) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aß present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer's disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aß isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aß. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aß, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aß molecule from zinc-mediated interactions with the same site of another Aß molecule can effectively inhibit the oligomerization and aggregation of Aß. Taking into account the similarity in the structural organization of zinc-binding sites within Aß and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aß. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aß and Aß with isomerized Asp7 (isoAß). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aß(1-42) and isoAß(1-42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aß in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

"Suppressor Factor" of Neutrophils: A Short Story of a Long-Term Misconception.

A large body of evidence obtained during the last decade has demonstrated that neutrophils suppress T cell proliferation in different models of inflammation and cell interaction. The commonly used method for assessing cell proliferation and proliferation inhibition is measuring [3H]thymidine incorporation into cells. Earlier, we observed inhibition of [3H]thymidine uptake in experiments on neutrophil-mediated regulation of T cell response in tuberculosis immunity. Here, we used different types of proliferating cells to analyze the nature of the soluble "neutrophil factor" by a variety of methods (dialysis, HPLC, mass spectrometry, and NMR) and unambiguously demonstrated that neutrophils do not synthesize a specific factor inhibiting cell proliferation, but secrete high concentrations of extracellular thymidine that competitively inhibit [3H]thymidine incorporation. Although the physiological significance of thymidine secretion by neutrophils remains unknown, this phenomenon should be carefully considered when designing test systems for studying cell-cell interactions.

The English (H6R) familial Alzheimer's disease mutation facilitates zinc-induced dimerization of the amyloid-ß metal-binding domain.

Interaction of Zn(2+) with the metal-binding domain of the English (H6R) amyloid-ß mutant results in the formation of peptide dimers. The mutation causes the exclusion of His6 from the zinc chelation pattern observed in the intact domain and triggers the assembly of the dimers via zinc ions coordinated by (11)EVHH(14) fragments.

NMR studies of ligand carboxylate group interactions with arginine residues in complexes of Lactobacillus casei dihydrofolate reductase with substrates and substrate analogues.

In a series of complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with substrates and substrate analogues, the (1)H/(15)N NMR chemical shifts for the guanidino group of the conserved Arg 57 residue were found to be sensitive to the mode of binding of their H(eta) protons to the charged oxygen atoms in ligand carboxylate groups. In all cases, Arg 57 showed four nonequivalent H(eta) signals indicating hindered rotation about the N(epsilon)-C(zeta) and C(zeta)-N(eta) bonds. The H(eta)(12) and H(eta)(22) protons have large downfield shifts as expected for a symmetrical end-on interaction with the ligand carboxylate group. The chemical shifts are essentially the same in the complexes with folate and p-aminobenzoyl-L-glutamate (PABG) and similar to those found previously for the methotrexate complex reflecting the strong and similar hydrogen bonds formed with the carboxylate oxygens. Interestingly, the rates of rotation about the N(epsilon)-C(zeta) bond for the complexes containing the weakly binding PABG fragment are almost identical to those measured in the complex with methotrexate, which binds 10(7) times more tightly. In the methotrexate complex, this rotation depends on correlated rotations about the N(epsilon)-C(zeta) bond of Arg 57 and the C(alpha)-C' bond of the ligand glutamate alpha-carboxylate group. Thus, even in a fragment such as PABG, which has a much faster off-rate, the carboxylate group binds to the enzyme in a similar way to that in a parent molecule such as folate and methotrexate with the rotation about the N(epsilon)-C(zeta) bond of Arg 57 being essentially the same in all the different complexes.

Validation of a new restraint docking method for solution structure determinations of protein-ligand complexes.

A new method is proposed for docking ligands into proteins in cases where an NMR-determined solution structure of a related complex is available. The method uses a set of experimentally determined values for protein-ligand, ligand-ligand, and protein-protein restraints for residues in or near to the binding site, combined with a set of protein-protein restraints involving all the other residues which is taken from the list of restraints previously used to generate the reference structure of a related complex. This approach differs from ordinary docking methods where the calculation uses fixed atomic coordinates from the reference structure rather than the restraints used to determine the reference structure. The binding site residues influenced by replacing the reference ligand by the new ligand were determined by monitoring differences in 1H chemical shifts. The method has been validated by showing the excellent agreement between structures of L. casei dihydrofolate reductase trimetrexate calculated by conventional methods using a full experimentally determined set of restraints and those using this new restraint docking method based on an L. casei dihydrofolate reductase methotrexate reference structure.

Structure and dynamics in solution of the complex of Lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate.

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N-labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein-ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 degrees C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower-temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4-diaminopyrimidine-containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side-chain orientations and local conformations of which are not greatly changed in the different complexes (similar chi1 and chi2 values).

Characterization of rates of ring-flipping in trimethoprim in its ternary complexes with Lactobacillus casei dihydrofolate reductase and coenzyme analogues.

NMR measurements have been used to investigate rates of ring-flipping and the activation parameters for the trimethoxybenzyl ring of the antibacterial drug trimethoprim (TMP) bound to Lactobacillus casei dihydrofolate reductase (DHFR) for a series of ternary complexes formed with analogues of the coenzyme NADPH. Rates were obtained at several temperatures from line shape analyses ((13)C-edited HSQC (1)H spectra) and transfer of magnetization measurements (zz-HSQC) on complexes containing 3'-O-[(13)C]trimethoprim. Examination of the structures of the complexes indicates that ring-flipping can only be achieved following major conformational changes and transient fluctuations of the protein and coenzyme structure around the trimethoxybenzyl ring. There is no simple correlation between rates of ring-flipping and binding constants. The presence of the coenzyme nicotinamide ring (in either its reduced or its oxidized forms) in the binding site close to the trimethoxybenzyl ring moiety is the major factor reducing the ring-flipping on coenzyme binding. Thus, the ternary complex with NADPH shows the largest reduction in the rate of ring-flipping (11 +/- 3 s(-)(1) at 298 K) as compared with the binary complex (793 +/- 80 s(-)(1) at 298 K). Complexes with NADPH analogues that either have no nicotinamide ring or are known to have their nicotinamide rings removed from the binding site show the smallest reductions. For the DHFR.TMP.NADP(+) complex where there are two conformations present, very different rates of ring-flipping were observed for the two forms. The activation parameters (DeltaH() and DeltaS()) for the ring-flipping in all the complexes are discussed in terms of the protein-ligand interactions and the possible constraints on the pathway through the transition state.

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate.

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.

High-resolution solution structure of human pNR-2/pS2: a single trefoil motif protein.

pNR-2/pS2 is a 60 residue extracellular protein, which was originally discovered in human breast cancer cells, and subsequently found in other tumours and normal gastric epithelial cells. We have determined the three-dimensional solution structure of a C58S mutant of human pNR-2/pS2 using 639 distance and 137 torsion angle constraints obtained from analysis of multidimensional NMR spectra. A series of simulated annealing calculations resulted in the unambiguous determination of the protein's disulphide bonding pattern and produced a family of 19 structures consistent with the constraints. The peptide contains a single "trefoil" sequence motif, a region of about 40 residues with a characteristic sequence pattern, which has been found, either singly or as a repeat, in about a dozen extracellular proteins. The trefoil domain contains three disulphide bonds, whose 1-5, 2-4 and 3-6 cysteine pairings form the structure into three closely packed loops with only a small amount of secondary structure, which consists of a short alpha-helix packed against a two-stranded antiparallel beta-sheet. The structure of the domain is very similar to those of the two trefoil domains that occur in porcine spasmolytic polypeptide (PSP), the only member of the trefoil family whose three-dimensional structure has been previously determined. Outside the trefoil domain, which forms the compact "head" of the molecule, the N and C-terminal strands are closely associated, forming an extended "tail", which has some beta-sheet character for part of its length and which becomes more disordered towards the termini as indicated by (15)N{(1)H} NOEs. We have considered the structural implications of the possible formation of a native C58-C58 disulphide-bonded homodimer. Comparison of the surface features of pNR-2/pS2 and PSP, and consideration of the sequences of the other human trefoil domains in the light of these structures, illuminates the possible role of specific residues in ligand/receptor binding.

NMR detection of arginine-ligand interactions in complexes of Lactobacillus casei dihydrofolate reductase.

1H-NMR and 15N-NMR signal assignments have been made for the eight arginine residues in Lactobacillus casei dihydrofolate reductase in its binary complex with methotrexate and in its ternary complex with methotrexate and NADPH. 1H-NMR chemical shifts for the guanidino groups of two of the arginines (Arg57 and Arg43) were sensitive to different modes of binding of the guanidino groups with charged oxygen atoms of the ligands. In the complexes formed with methotrexate, Arg57 showed four non-equivalent NH eta proton signals indicating hindered rotation about the N epsilon-C zeta and C zeta-N eta bonds. The NH eta 12 and NH eta 22 protons showed large downfield shifts, which would be expected for a symmetric end-on interaction of these protons with the charged oxygen atoms of a carboxylate group in methotrexate. These effects were not observed for the complex formed with trimethoprim, which does not contain any carboxylate groups. In the complex formed with NADPH present, Arg43 showed a large downfield chemical shift for its NH epsilon proton and a retardation of its rate of exchange with water. This pattern of deshielding contrasts with that detected for Arg57 and is that expected for a side-on interaction of the guanidino group protons with charged oxygen atoms of the ribose 2'-phosphate group of NADPH.

NMR-based structural studies of the pNR-2/pS2 single domain trefoil peptide. Similarities to porcine spasmolytic peptide and evidence for a monomeric structure.

NMR spectroscopy measurements have been used to obtain structural information about the pNR-2/pS2 single-domain trefoil peptide. NMR data from 2D (two dimensional) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), NOE spectroscopy (NOESY), rotating frame NOE spectroscopy (ROESY) and 2D 13C-1H heteronuclear single-quantum coherence (HSQC) and 13C-1H HSQC-TOCSY spectra have been analysed to provide essentially complete 1H and 13C sequence-specific assignments for the pNR-2/pS2 protein. From a consideration of the NOE intensities, 3J(NH-alpha CH) coupling constants, 1H and 13C chemical shifts of backbone atoms and amide-proton exchange rates, the pNR-2/pS2 was found to contain two short antiparallel beta-strands (32-35 and 43-46), a short helix (25-30) and a type I beta-turn (11-15). These elements of secondary structure are very similar to those found in the two trefoil domains of pSP for which detailed structural information is already available. Similar 1H chemical shifts were noted for several conserved residues in pNR-2/pS2 and pSP and a characteristic Phe residue with a slowly flipping ring was found in the pNR-2/pS2 variant and in both domains of pSP. The tertiary structures of the domains therefore appear to be very similar in the two proteins and it is likely that the pNR-2/pS2 has the same pattern of disulphide bonds (1-5, 2-4, 3-6) as pSP. Correlation time measurements derived from 1H-1H NOE measurements indicate that the Cys58-->Ser form of the pNR-2/pS2 protein used in this study is monomeric in solution at approximately 2 mM.

Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase.

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate.

Determination of stereospecific assignments, torsion-angle constraints, and rotamer populations in proteins using the program AngleSearch.

A general program, AngleSearch, which calculates coupling constants and interproton distances for any molecular fragment and does a grid search to find torsion angles, rotamer populations, and stereospecific assignments which fit the measured data has been developed. The program takes full advantage of the fact that ratios of cross-peak intensities (measured in HNHB and HN(CO)HB experiments) can provide accurate ratios of coupling constants even for large molecules. AngleSearch is capable of: (a) analyzing any type of residue including protein, RNA, DNA, and ligand residues; (b) conformational grid searching in dihedral-angle space using 6 degree steps; (c) averaging coupling constants and (l/r6) distances for rotamers undergoing fast exchange; (d) grid or Monte Carlo searching for populations of staggered rotamers; (e) using all available distance-related data from ROESY and/or NOESY spectra; (f) using any available coupling constant data having known relationships to corresponding dihedral angles; and (g) directly using cross-peak intensities related to values of coupling constants. The program can also assist in the stereospecific assignment of the alpha-CH2 protons of glycine residues. The effects of the quality of the input data on the results of the AngleSearch calculations have been assessed.

Solution structure of bound trimethoprim in its complex with Lactobacillus casei dihydrofolate reductase.

Two- and three-dimensional (2D and 3D) NMR techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (M(r) 18,300) in its 1:1 complex with the antibacterial drug trimethoprim. A sample of uniformly 15N-labeled protein was examined using 3D 15N/1H experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOEs between trimethoprim and protein protons and four intramolecular NOEs in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15N-and 13C-labeled trimethoprim molecules ([1,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound to unlabeled protein. The ligand-protein NOEs were used as distance constraints in conjunction with minimum energy and simulated annealing calculations (carried out with X-PLOR) to dock the trimethoprim ligand into dihydrofolate reductase, using as a starting structure the crystal coordinates from a related complex with a similar overall protein structure. The restrained minimum energy calculations and the simulated annealing calculations gave 83 calculated structures with distance violations of < 0.1 A. In all of these, the two aromatic rings of trimethoprim occupied essentially the same region of conformational space in the binding site (RMSD = 0.63 A). The protein residues nearest to the bound trimethoprim were found to be very similar in all of the structures and agreed well with corresponding contact residues observed in the X-ray crystal studies on trimethoprim complexes formed with Escherichia coli and chicken liver DHFRs.

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution.

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.