Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate .cntdot. NADP+ ternary complex. substrate binding and a model for the transition state

Bystroff, Christopher; Oatley, Stuart J.; Kraut, Joseph

doi:10.1021/bi00465a018

Cited by 274 publications

(375 citation statements)

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“…This is in contrast to the adenosine moiety for both NADPH and NADP', where shifts in their respective Raman spectra clearly show the result of protein interactions. These data are in general agreement with X-ray results (Bystroff et al, 1990) which show the oxidized nicotinamide ring in binary complex with DHFR to be disordered, suggesting that the group may reside in solution, while the electron density patterns for the rest of the cofactor are well defined and place the adenosine moiety in its protein binding pocket. This may be a result from there being no compensating polar groups at the nicotinamide binding pocket which could stabilize the charge on the positive NADP' (Bystroff et al, 1990).…”

Section: Resultssupporting

confidence: 88%

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A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy

Zheng

Chen

Callender

1993

European Journal of Biochemistry

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We report here the Raman spectra of NADPH, NADP', 3-acetylpyridine adenine dinucleotide (AcPdADP'), NADH and a fragment of these molecules, 2'-phospho-adenosine-5'-diphosphoribose (Ado%'pS'ppRib), bound to Escherichia coli dihydrofolate reductase (DHFR). The positions that are observed for the bound adenosine 'triplet' bands are consistent with a protein binding pocket for this group which is quite hydrophobic in nature. No binding effect is observed on Raman bands associated with the nicotinamide group of NADP' as a binary complex with DHFR, suggesting very loose, if any, binding of this group. In contrast, changes in the Raman spectrum of the nicotinamide group of NADP' bound to an inhibitor (trimethoprim) ternary complex of DHFR are clearly observed which indicate substantial binding interaction. The carboxamide group of bound NADPH (and NADH) adopts the trans conformation. A 35-cm-' upshift is observed in the rocking motion of the carboxamide -NH, group of NADPH, and a 5-cm-' upward shift is seen in the C=O stretch mode of AcPdADP' upon binding to the enzyme-trimethoprim complex. These results suggest that the -NH, group of the carboxamide moiety is more tightly hydrogen bonded in the protein binding pocket than in solution while that of the C=O group is less tightly hydrogen bonded; these hydrogen bonds would appear to be responsible for holding the nicotinamide headgroup in place properly for catalysis. We have compared this with the results obtained previously in other protein complexes, and interpret the observed shifts in these bands as a measure of the hydrogen bonding enthalpy of the -NH, and C =O groups with their protein environments. Perhaps surprisingly, the magnitude of the hydrogen bonding enthalpy takes on a limited number of discrete values over five protein complexes rather than over a continuous range. The effect that this has on the catalytic properties of DHFR and the other NAD dehydrogenases that we have studied to date, particularly their stereochemistry, is discussed. A small downward shift is observed for the P -0 stretch of the 2'-phosphate moiety of NADP. This indicates that the 2'-phosphate moiety binds to DHFR in the dianionic form. Futhermore, the local enthalpic interaction that the 2'-phosphate group has with protein is stronger than its interaction with water.

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Section: Resultssupporting

confidence: 88%

“…DHFR from E. coli was prepared as previously described (Bystroff et al, 1990) and stored at 4°C in 10 mM TridHCl, pH 8.0, 1 mg/ml 0.5 M KCI. Before use, insoluble protein was removed by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy

Zheng

Chen

Callender

1993

European Journal of Biochemistry

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“…NAD(P) binding to dehydrogenases has been studied in detail [ 11,121. It was shown that upon binding of the coenzyme, alcohol dehydrogenases undergo a large conformational change: the catalytic domain makes a large rigid-body rotation towards the coenz~e-binding domain. Similar studies have been performed for NADP+ in dihydrofolate reductase [13], the thiamin diphosphate dependent enzyme transketolase 1141, and other enzymes [15].…”

Section: Introductionsupporting

confidence: 61%

“…First, residues from regions which have ill-defined electron density (residues 19,137,200,330). Second, residues from intersubunit contact areas (residues 13,19,87). Third, residues from flexible loops near the flavin binding site.…”

Section: Structure Of Apo-enzymementioning

confidence: 99%

Crystal structure of apo‐glycolate oxidase

Sandalova

Lindqvist

1993

FEBS Letters

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The crystal structure of the apoform of the a&barrel enzyme glycoiate oxidase has been determined to 2.6 A resolution. Removal of the tightly bound cofactor FMN has a very strong influence on the protein structure; it is converted into a very flexible state, verging on a molten globule type of structure. The asymmetric unit contains two subunits with different conformations to each other and to the halo-enzyme. The secondary structures are preserved, but their mutual arrangement has changed to some extent introducing cavities into the protein. The largest structural shifts are, however, found in the loops.

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“…The apo enzyme is difficult to crystallize (Bystroff & Kraut, 1991), so we chose to examine the binary complex with the inhibitor methotrexate. Methotrexate binds in the folate-binding site and leaves the NADP site empty Filman et al, 1982;Bystroff et al, 1990;Bystroff & Kraut, 1991). To create the binary complex, 20 mg of apo enzyme was dialyzed against 0.1 M Tris buffer (pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

The effect of denaturants on protein structure

et al. 1997

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Virtually all studies of the protein-folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X-ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. J Mol B i d 226491-505).Keywords: denaturants; dihydrofolate reductase; protein mobility; ribonuclease A Despite our routine use of them, our understanding of the molecular mechanism by which chemical denaturants like urea or guanidinium (Gua) cause a protein to unfold is still rather limited. As has been pointed out by others (Schellman, 1987), part of the problem is that it is difficult to measure binding constants when the protein concentration is M and the denaturant concentration is 4 M. High precision scanning calorimetry has provided one experimental solution to this problem (Makhatadze & Privalov, 1992) but relating the thermodynamic data produced in such an experiment to protein structure can be difficult (Alber, 1989). While it does seem clear that denaturants interact directly with the protein (Simpson & Kauzmann, 1953), it is also clear that at high concentrations these molecules can cause substantial changes to the behavior of the solvent itself (Breslow & Guo, 1990 teins by migrating into the interior of the protein and forming hydrogen bonds to atoms in the backbone (Hedwig et al., 1991). In support of such a model, the exchange rates of some peptide NH protons, which are found on both the surface and the interior of a protein, are decreased in the presence of urea (Kim & Woodward, 1993). In addition, crystallographic studies of diketopiperazine and urea show hydrogen bonding between the amide groups and the urea (Thayer et al., 1993). Other models have suggested that denaturants act by decreasing the hydrophobic effect (Wetlaufer et al., 1964;Roseman & Jencks, 1975), which, in this instance, is usually postulated to be the dominant force stabilizing protein structure. Finally, experiments by Kim and Woodward (1 993) clearly show that, in addition to interacting with peptide bonds, urea alters...

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Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate .cntdot. NADP+ ternary complex. substrate binding and a model for the transition state

Cited by 274 publications

References 39 publications

A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy

A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy

Crystal structure of apo‐glycolate oxidase

The effect of denaturants on protein structure

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