The charge state of trimethoprim bound to <i>Lactobacillus casei</i> dihydrofolate reductase

Roberts, G. C. K.; Feeney, J.; Burgen, A. S. V.; Daluge, Susan M.

doi:10.1016/0014-5793(81)80893-9

Cited by 43 publications

(34 citation statements)

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“…With the advent of structural information on trimethoprim binding to the enzyme, both from crystallography (Baker et al, 1981(Baker et al, , 1983Matthews et al, 1983;Stammers et al, 1983) and from NMR spectroscopy (Birdsall et al, 1977Cayley et al, 1979;Gronenborn et al, 1981a,b,c;Roberts et al, 1981;Roberts, 1983a), it has become possible to begin to interpret these relationships in molecular detail [see, e.g., Roth & Cheng (1982), Li et al (1982), Baker et al (1983), Matthews et al (1983), Birdsall et al (1983), Roberts (1983a), and Stuart et al (1983)l.…”

mentioning

confidence: 98%

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP complex

Birdsall¹,

Bevan²,

Pascual³

et al. 1984

Biochemistry

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The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.

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mentioning

confidence: 98%

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP complex

Birdsall¹,

Bevan²,

Pascual³

et al. 1984

Biochemistry

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“…Cannon and co-workers [121] have predicted the pK a value for the N1 position in the DHFR ± methotrexate complex and their quantum chemistry calculations suggest that N1 remains unprotonated and forms a hydrogen bond with an unionized carboxylate group on the conserved Asp residue, which is in contradiction to the experimental results. While the experimental evidence for N1 protonation appears to be strong, the definitive experiment of examining the DHFR complex with [1-15 N]labeled methotrexate has not yet been carried out: a protonated N1 would be detected as a 15 [92,122] Figure 12 shows that the 13 C chemical shift of the bound species is similar to that of the free protonated N1 species and the pK of the bound trimethoprim is decreased by at least 3 pK units.…”

Section: Protein Interactions Involving the Pyrimidine Ring Of The Inmentioning

confidence: 93%

NMR Studies of Ligand Binding to Dihydrofolate Reductase

Feeney¹

2000

Angewandte Chemie International Edition

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Unique capabilities are offered by NMR spectroscopy for probing specific interactions of enzymes with substrates and substrate analogues, for characterizing the multiple conformations of the complexes, and for measuring rates of a wide range of dynamic processes. The most extensive studies of this type have been directed at complexes formed by dihydrofolate reductase with antifolate drugs and are described in this review.

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“…As an extreme example, Egsite predicts that 13 should bind at least rather well, and possibly extraordinarily tightly; but from this explanation of this training set, it is impossible to be more precise. In our previous work, Egsets produced literally thousands of relatively narrowly specified site models, and searching through these for ones of high predictive power was a great challenge.…”

Section: Cocaine Analoguesmentioning

confidence: 96%

Intervals and the deduction of drug binding site models

Crippen

1995

J Comput Chem

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In the search for new drugs, it often occurs that the binding affinities of several compounds to a common receptor macromolecule are known experimentally, but the structure of the receptor is not known. This article describes an extraordinarily objective computer algorithm for deducing the important geometric and energetic features of the common binding site, starting only from the chemical structures of the ligands and their observed binding. The user does not have to propose a pharmacophore, guess the bioactive conformations of the ligands, or suggest ways to superimpose the active compounds. The method takes into account conformational flexibility of the ligands, stereospecific binding, diverse or unrelated chemical structures, inaccurate or qualitative binding data, and the possibility that chemically similar ligands may or may not bind to the receptor in similar orientations. The resulting model can be viewed graphically and interpreted in terms of one or more binding regions of the receptor, each preferring to be occupied by various sorts of chemical groups. The model always fits the given data completely and can predict the binding of any other ligand, regardless of chemical structure. The method is an outgrowth of distance geometry and Voronoi polyhedra site modeling but incorporates several novel features. The geometry of the ligand molecules and the site is described in terms of intervals of internal distances. Determining the site model consists of reducing the uncertainty in the interregion distance intervals, and this uncertainty is described as intervals of intervals. Similarly, the given binding affinities and their experimental uncertainties are treated as intervals in the affinity scale. The final site model specifies an entire region of interaction energy parameters that satisfy the training set rather than a single set of parameters. Predicted binding for test compounds results in an interval which, when compared to the experimental interval, may be correct, incorrect, or vague. There is a pervasive ternary logic involved in the assessment of predictions, in the search for a satisfactory model, and in judging whether a given molecule may bind in a particular orientation: true, false, or maybe. The approach is illustrated on an extremely simple artificial example and on a real data set of cocaine analogues binding to a nerve membrane receptor in vitro. 0 1995 by John Wiley & Sons, Inc.

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The charge state of trimethoprim bound to Lactobacillus casei dihydrofolate reductase

Cited by 43 publications

References 18 publications

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP complex

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP complex

NMR Studies of Ligand Binding to Dihydrofolate Reductase

Intervals and the deduction of drug binding site models

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