Dihydrofolate reductase: multiple conformations and alternative modes of substrate binding

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent [Birdsall, B., Gronenborn, A. M., Hyde, E. I., Clore, G. M., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1982) Biochemistry 21, 5831]. The assignment of the resonances of all the aromatic protons of the ligand molecules in all three conformational states of the complex has now been completed by using a variety of NMR methods, particularly two-dimensional exchange experiments. The resonances of the nicotinamide protons of the coenzyme and the pteridine 7-proton of the folate have different chemical shifts in the three conformations, in some cases differing by more than 1 ppm. Comparison of the COSY spectra of the complex at low pH (conformation I) and high pH (conformations IIa and IIb) with that of the enzyme-methotrexate-NADP+ complex shows only slight differences in the conformation of the protein. The pattern of chemical shift changes in the ligand and the protein indicates that the structural differences are localized within the active site of the enzyme. Nuclear Overhauser effects (NOEs) are observed between the nicotinamide 5- and 6-protons and the methyl resonance of Thr 45 at both low and high pH, indicating that there is no major movement of the nicotinamide ring. By contrast, NOEs are observed between the pteridine 7-proton and the methyl protons of Leu 19 and Leu 27 in conformations I and IIa but not in conformation IIb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional 1H NMR of gene 5 protein indicates that only two aromatic rings interact significantly with oligodeoxynucleotide bases

The interaction of gene 5 protein (G5P) with oligodeoxynucleotides is investigated by 1H NMR methods, principally two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Aromatic resonances of G5P are specifically assigned from crystallographic data, while the low-field resonances of nucleotides are assigned with sequential or other procedures. Chemical shift changes that accompany binding of d(pA)4, d(A)4, d(pT)4, and d(pA)8, combined with specific protein-nucleotide nuclear Overhauser effects (NOEs) obtained from NOESY spectra, suggest that Phe-73 and Tyr-26 are the only aromatic residues that stack significantly with nucleotide bases. Chemical shift data also imply a role for Leu-28, though this has not been confirmed with intermolecular NOEs. Binding of all four oligonucleotides causes marked upfield movements (0.1-0.6 ppm) of G5P NOESY cross peaks belonging to Tyr-26, Leu-28, and Phe-73. Most other G5P spin systems, notably those of Tyr-34 and Tyr-41, do not appear to be significantly affected. In the d(pA)4-G5P complex an intermolecular NOE is observed between Tyr-26 and H1' of Ade-1, while Phe-73 has NOEs with the H2, H8, and H1' protons of Ade-2 and -3. Intramolecular NOEs seem to follow a similar pattern in the partially cooperative d(pA)8-G5P complex, though specific nucleotide resonance assignments are not possible in this case. Binding causes relatively small chemical shift changes for the base resonances in adenylyl nucleotides, suggesting that there is some, but not complete, unstacking of the bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms. Resonance assignment, secondary structure and ligand binding.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

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.     

Identification of individual protein–ligand NOEs in the limit of intermediate exchange

Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations. Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we employ methods related to driven NOE spectroscopy to investigate protein–ligand interactions in the intermediate exchange regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples selectively protonated at a few sites so that the 1D ¹H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein–ligand NOEs that allow calculation of initial complex structures, suitable for structure-based optimization of primary drug leads obtained from high-throughput screening. The method was applied to measure individual intermolecular NOEs between the anti-apoptotic protein Bcl-xL at 25 μM and a “first generation” small-molecule ligand, for which the spectrum is entirely broadened at stoichiometric concentrations. This approach is general and can also be used to characterize protein–protein or protein–nucleic-acid complexes.     

Partial 1H NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate

Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques. Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure. Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues. Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis. The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments. As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex. Under these experimental conditions, only one bound form of the pterin ring could be detected.     

Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin.

The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate). The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine [Volz, K. W., Matthews, D. A., Alden, R. A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring. This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring. Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates. The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277], suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes. Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly. A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release.     

Some NMR Experiments and a Structure Determination Employing a {15N,2H} Enriched Protein

We present the results of studies of an aqueous sample of a highly {15N,2H} enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7Å apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.     

Two-dimensional 1H-NMR studies of horseradish peroxidase C and its interaction with indole-3-propionic acid

The binding of aromatic donor molecules to plant peroxidases has been investigated by examining the complex formed between horseradish peroxidase isoenzyme C and indole-3-propionic acid using two-dimensional 1H-NMR spectroscopy. Despite the relatively high molecular mass and paramagnetism of the protein, this technique can be successfully applied to provide new information on the structure of the complex. A number of relatively well-resolved resonances in certain regions of the one-dimensional spectrum are assigned to amino acid type on the basis of the two-dimensional experiments. Two phenylalanine side chains are found to interact at positions close to the haem group as shown by nuclear Overhauser effect spectroscopy (NOESY). Furthermore, the NOESY spectrum of the complex reveals distinct interactions between these phenylalanine residues and the indole ring of the donor molecule. The binding site is found to comprise of these phenylalanine side chains and also the methyl group of a leucine or valine residue. On the basis of the model structure of horseradish peroxidase isoenzyme C proposed by Welinder and N?rskov-Lauritsen and information from previous studies of the related turnip peroxidases, possible locations for this binding site are discussed. The NMR methods adopted here may be generally applicable to the study of peroxidase--aromatic-donor interactions.     

Sequential 1H NMR assignments of iron(II) cytochrome c551 from Pseudomonas aeruginosa

Sequence-specific 1H NMR resonance assignments for all but the C-terminal Lys 82 are reported for iron(II) cytochrome c551 from Pseudomonas aeruginosa at 25 degrees C and pH = 6.8. Spin systems were identified by using TOCSY and DQF-COSY spectra in 2H2O and 1H2O. Sequential assignments were made by using NOESY connectivities between adjacent amide, alpha, and beta protons. Resonances from several amino acids including His 16, Gly 24, Ile 48, and Met 61 experience strong ring-current shifts due to their placement near the heme. All heme protons, including the previously unassigned propionates, have been identified. Preliminary analysis of sequential and medium-range NOEs provides evidence for substantial amounts of helix in the solution structure. Long-range NOEs indicate that the folds in solution and crystal structures are similar. For one aromatic side chain (Tyr 27) that is close to the heme group we found a transition from hindered ring rotation at low temperature to rapid rotation at high temperature.     

Application of automated NOE assignment to three-dimensional structure refinement of a 28 kDa single-chain T cell receptor

An automated procedure for NOE assignment and three-dimensional structure refinement is presented. The input to the procedure consists of (1) an ensemble of preliminary protein NMR structures, (2) partial sequence-specific assignments for the protein and (3) the positions and volumes of unassigned NOESY cross peaks. Chemical shifts for unassigned side chain protons are predicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment and structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 408–422]. ARIA is optimized for the task of structure refinement of larger proteins. Errors are filtered to ensure that sequence-specific assignments are reliable. The procedure is applied to the 27.8 kDa single-chain T cell receptor (scTCR). Preliminary NMR structures, nearly complete backbone assignments, partial assignments of side chain protons and more than 1300 unassigned NOESY cross peaks are input. Using the procedure, the resonant frequencies of more than 40 additional side chain protons are assigned. Over 400 new NOE cross peaks are assigned unambiguously. Distances derived from the automatically assigned NOEs improve the precision and quality of calculated scTCR structures. In the refined structures, a hydrophobic cluster of side chains on the scTCR surface that binds major histocompatibility complex (MHC)/antigen is revealed. It is composed of the side chains of residues from three loops and stabilizes the conformation of residues that interact with MHC.     

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.     

A 500 MHz proton NMR study of stacking interactions: binding of tripeptide Lys-Tyr-Lys to tetradeoxynucleotide d-GpCpGpC.

The complete sequential assignment and conformation of d-GpCpGpC in D2O has been determined from 1D NMR spectra at 285-320 K and room temperature 2D-COSY and NOESY spectra. The tetradeoxynucleotide exists primarily as a right handed double helix at 285 K, having Tm as 314 K. On binding to a tripeptide Lys-Tyr-Lys in a concentration equimolar to tetranucleotide duplex, the Tyr ring protons shift upfield by 0.14 ppm at 285 K. The increase in Tm on binding suggests stabilization of duplex. The existence of intermolecular NOEs between C4 sugar protons and Tyr alpha C and Lys alpha C protons give direct evidence of proximity of Tyr residue to the C4 base of d-GpCpGpC. The conformation of d-GpCpGpC remains unchanged on binding. The observed results are interpreted in terms of preferential stacking of aromatic ring of Tyr residue with proximal base-pair of d-GpCpGpC, stabilized by electrostatic interaction of Lysine side chains with backbone phosphates. This is in contrast to intercalculation of aromatic dyes within base-pairs resulting in a change in sugar conformation at the binding site.     

1H NMR studies on the interaction between distamycin A and a symmetrical DNA dodecamer

High-resolution NMR techniques have been used to examine the structural and dynamical features of the interaction between distamycin A and the self-complementary DNA dodecamer duplex d-(CGCGAATTCGCG)2. The proton resonances of d(CGCGAATTCGCG)2 have been completely assigned by previous two-dimensional NMR studies [Hare, D. R., Wemmer, D. E., Chou, S. H., Drobny, G., & Reid, B. R. (1983) J. Mol. Biol. 171, 319-336]. Addition of the asymmetric drug molecule to the symmetric dodecamer leads to the formation of an asymmetric complex as evidenced by a doubling of DNA resonances over much of the spectrum. In two-dimensional exchange experiments, strong cross-peaks were observed between uncomplexed DNA and drug-bound DNA resonances, permitting direct assignment of many drug-bound DNA resonances from previously assigned free DNA resonances. Weaker exchange cross-peaks between formerly symmetry related DNA resonances indicate that the drug molecule flips head-to-tail on one duplex with half the frequency at which it leaves the DNA molecule completely. In experiments performed in H2O, nuclear Overhauser effects (NOEs) were observed from each drug amide proton to an adenine C2H and a pyrrole H3 ring proton. In two-dimensional nuclear Overhauser experiments performed on D2O solutions, strong intermolecular NOEs were observed between each of the three pyrrole H3 resonances of the drug and an adenine C2H resonance, with weaker NOEs observed between the drug H3 resonances and C1'H resonances. The combined NOE data allow us to position the distamycin A unambiguously on the DNA dodecamer, with the drug spanning the central AATT segment in the minor groove.     

Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's beta-amyloid precursor protein.

Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.     

Structural comparisons of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz

We have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring [the 3',5'-difluoro and 3',5'-dichloro analogues (II and III)] and also the glutamic acid moiety [the alpha- and gamma-monoamides (IV and V)]. Assignments of the 1H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those of complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes (15 degrees and 25 degrees in the difluoro- and dichloromethotrexate complexes, respectively). Binding of oxidized or reduced coenzyme (NADP+ or NADPH) to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.     

Sequence specific molecular recognition by a monocationic lexitropsin of the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

All 1H-NMR resonances of d-[CATGGCCATG]2 and the 1:1 complex of lexitropsin 1 and the DNA were assigned by the NOE difference, COSY and NOESY methods. Addition of 1 causes the base and imino protons for the sequence 5'-CCAT to undergo the most marked drug-induced chemical shift changes, thereby indicating that 1 is located in this base pair sequence. NOEs confirmed the location and orientation of the drug in the 1:1 complex, with the amino terminus oriented to C(6). The van der Waals interaction between H12a,b of 1 and AH2(8) may be responsible for reading of the 3' A.T base pair in the 5'-CCAT sequence. Exchange NMR effects allow an estimate of approximately equal to 62 s-1 for the intramolecular "slide-swing" exchange of the lexitropsin between two equivalent binding sites with delta G = 58 +/- 5 kJ mol-1 at 301 degrees K.     

Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus

The nuclear Overhauser effects (NOEs) observed between heme substituent protons and a small number of nearby protein side chain protons in the water-elimination Fourier transform NOE spectroscopy (WEFT-NOESY) spectra of high- and low-spin wild-type nitrophorin (NP) 2 and its ligand complexes have been analyzed and compared with those observed for the same complexes of wild-type NP3. These assignments were made on naturally abundant isotope samples, with the most useful protein side chains being those of Ile120, Leu122, and Leu132 for NP2 and NP3, and Thr121, Leu123, and Leu133 for NP1 and NP4. It is found that the NOEs observed are identical, with extremely similar protein side chain proton chemical shifts. This is strong evidence that the structure of NP3, for which no X-ray crystal structures are available, is essentially identical to that of NP2, at least near the heme binding pocket. Similarly, the NOEs observed between heme substituents and protein side chains for NP1 and NP4 also indicate that the structures of the protein having both A and B heme orientations are very similar to each other, as well as to the proteins with major B heme orientation of NP2 and NP3. These A and B connectivities can be seen, even though the two heme orientations have similar populations in NP1 and NP4, which complicates the analysis of the NOESY spectra. The histamine complex of wild-type NP2 shows significant shifts of the Leu132 side chain protons relative to all other ligand complexes of NP1-NP4 because of the perturbation of the structure near Leu132 caused by the histamine's side chain ammonium hydrogen bond to the Asp29 side chain carboxylate.