Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Osborne, Michael J.; Venkitakrishnan, Rani P.; Dyson, H. Jane; Wright, Peter E.

doi:10.1110/ps.03219603

Cited by 38 publications

(48 citation statements)

References 35 publications

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“…Although the holoenzyme has been crystallized in both closed and occluded states as shown in Figure 1B, recent solution NMR work shows it to be predominantly closed in solution (Osborne et al, 2003). The MTX complex, considered a model for DHFR’s transition state, is also found in the closed conformation (Matthews et al, 1977; Sawaya and Kraut, 1997).…”

Section: Resultsmentioning

confidence: 99%

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

2009

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Summary The arduous task of rationally designing small molecule enzyme inhibitors is complicated by the inherent flexibility of the protein scaffold. To gain insight into the changes in dynamics associated with small molecule based inhibition, we have characterized, using NMR spectroscopy, E. coli dihydrofolate reductase in complex with two drugs: methotrexate and trimethoprim. The complexes allowed the intrinsic dynamic effects of drug binding to be revealed within the context of the “closed” structural ensemble. Binding of both drugs results in an identical decoupling of global motion on the micro- to millisecond timescale. Consistent with a change in overall dynamic character, the drugs’ perturbations to pico- to nanosecond backbone and side-chain methyl dynamics are also highly similar. These data show that the inhibitors simultaneously modulate slow concerted switching and fast motions at distal regions of DHFR, providing a dynamic link between the substrate binding site and distal loop residues known to affect catalysis.

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Section: Resultsmentioning

confidence: 99%

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

2009

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“…NMR data provide independent yardsticks, but not necessarily a one-to-one correspondence to molecular flexing. Chemical shift (CS) perturbations reflect changes in the local chemical environment, including, but not limited to conformation change or direct influence of ligand-binding (Osborne et al, 2003; Zuiderweg, 2002). NMR relaxation exchange contributions (R ex ), quantified from relaxation dispersion experiments, reflects dynamic changes in CS occurring on μs-to-ms time scales (Boehr et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Parsimony in Protein Conformational Change

et al. 2015

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Summary Protein conformational change is analyzed by finding the minimalist backbone torsion angle rotations that superpose crystal structures within experimental error. Of several approaches to enforcing parsimony during flexible least-squares superposition, an ℓ1-norm restraint provided greatest consistency with independent indications of flexibility from NMR relaxation dispersion and chemical shift perturbation in arginine kinase, and four previously studied systems. Crystallographic cross-validation shows that the dihedral parameterization describes conformational change more accurately than rigid-group approaches. The rotations that superpose the principal elements of structure constitute a small fraction of the raw (φ, ψ)-differences that also reflect local conformation and experimental error. Substantial long-range displacements can be mediated by modest dihedral rotations, accommodated even within α-helices and β-sheets without disruption of hydrogen bonding at the hinges. Consistency between ligand-associated and intrinsic motions (in the unliganded state) implies that induced changes tend to follow low-barrier paths between conformational sub-states that are in intrinsic dynamic equilibrium.

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“…Solution structures of the binary and ternary LcDHFR enzyme complexes are also available (1, 2, 22, 23), though no analysis of shift differences between binary and ternary enzyme complexes has been previously reported. Comparison of the NADPH-bound binary complex (PDB ID 2HQP) and the trimethoprim – NADPH-bound ternary complex (PDB ID 1LUD (1)) shows the greatest structural differences in regions already indicated by the EcDHFR NMR studies (4, 5) and our own BaDHFR NMR studies (Figure S1 in Supporting Information shows a superposition of the binary and ternary LcDHFR structures and notes the structural differences). A superposition of the three structures (BaDHFR, EcDHFR, and LcDHFR) yields a structural alignment (Figure 6) and facilitates the interpretation of residue characteristics at equivalent positions.…”

Section: Resultsmentioning

confidence: 71%

“…For BaDHFR, differences were interpreted from the titration HSQC data shown in Figure 4. For EcDHFR, differences were taken from refs 4 and 5. For LcDHFR, differences were interpreted from comparisons between the reported binary and ternary structures.…”

Section: Figures and Tablesmentioning

confidence: 99%

The Solution Structure of Bacillus anthracis Dihydrofolate Reductase Yields Insight into the Analysis of Structure−Activity Relationships for Novel Inhibitors

et al. 2009

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There is a significant need for new therapeutics to treat infections caused by the biodefense agent Bacillus anthracis. In pursuit of drug discovery against this organism, we have developed novel propargyl-linked inhibitors that target the essential enzyme dihydrofolate reductase (DHFR) from B. anthracis. Previously, we reported an initial series of these inhibitors and a high-resolution crystal structure of the ternary complex of the enzyme bound to its cofactor and one of the most potent inhibitors, UCP120B [Beierlein, J., Frey, K., Bolstad, D., Pelphrey, P., Joska, T., Smith, A., Priestley, N., Wright, D., and Anderson, A. (2008) J. Med. Chem. 51, 7532-7540]. Herein, we describe a threedimensional solution structure of the ternary complex as determined by NMR. A comparison of this solution structure to the crystal structure reveals a general conservation of the DHFR fold and cofactor interactions as well as differences in the location of an active site helix and specific ligand interactions. In addition to data for the fully assigned ternary complex, data for the binary (enzymecofactor) complex were collected, providing chemical shift comparisons and revealing perturbations in residues that accommodate ligand binding. Dynamics of the protein, measured using 15 N T 1 and T 2 relaxation times and { 1 H}-15 N heteronuclear NOEs, reveal residue flexibility at the active site that explains enzyme inhibition and structure-activity relationships for two different series of these propargyl-linked inhibitors. The information obtained from the solution structure regarding active site flexibility will be especially valuable in the design of inhibitors with increased potency.Bacillus anthracis, the causative agent of anthrax, is a well-known bioterrorism threat. The limited number of approved therapeutics exhibit serious drawbacks including indication spectrum, resistance, and expense, especially in a case of large-scale exposure. The development of stable, effective new therapeutics is certainly warranted.Dihydrofolate reductase (DHFR), 1 an essential enzyme in cellular metabolism, catalyzes the reduction of di-hydrofolate to form tetrahydrofolate using the cofactor NADPH. Over the past five decades, DHFR has been recognized as a validated drug target for both human ‡ Coordinates have been deposited in the Protein Data Bank with accession code 2KGK. † Funding provided by NIH Grant R01AI073375 (to A.C.A.). Figure S1 showing a superposition of the binary and ternary LcDHFR structures with structural differences noted and tables of chemical shift assignments for NADPH (Table S1) and UCP120B (Table S2) as well as a table of NOE signals involving helix B and the ligand (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org. In general, the solution structures are similar to the related X-ray crystal structures and yield additional details regarding the cooperativity of cofactor and ligand binding. Specifically, the solution structures of LcDHFR and hDHFR bound to NADPH and tri...

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Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Abstract: Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution.

Cited by 38 publications

References 35 publications

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

Parsimony in Protein Conformational Change

The Solution Structure of Bacillus anthracis Dihydrofolate Reductase Yields Insight into the Analysis of Structure−Activity Relationships for Novel Inhibitors

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