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.
Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.
Structure-based drug design relies on static protein structures despite significant evidence for the need to include protein dynamics as a serious consideration. In practice, dynamic motions are neglected because they are not understood well enough to model – a situation resulting from a lack of explicit experimental examples of dynamic receptor-ligand complexes. Here, we report high-resolution details of pronounced ~1 ms timescale motions of a receptor-small molecule complex using a combination of NMR and X-ray crystallography. Large conformational dynamics in Escherichia coli dihydrofolate reductase are driven by internal switching motions of the drug-like, nanomolar-affinity inhibitor. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments and NOEs revealed the crystal structure to contain critical elements of the high energy protein-ligand conformation. The availability of accurate, structurally resolved dynamics in a protein-ligand complex should serve as a valuable benchmark for modeling dynamics in other receptor-ligand complexes and prediction of binding affinities.
NMR chemical shifts of 1 H, 13 C, and 73 Ge, molecular modeling, and single-crystal X-ray diffraction results are reported for a series of substituted tris-and tetrakis(phenyl)germanes of the type (XC 6 H 4 ) 3 GeY and (XC 6 H 4 ) 4 Ge, where X = o-, m-, and p-OCH 3 , o-, m-, and p-OC 2 H 5 , m-and p-CF 3 , H, p-C(CH 3 ) 3 , p-Cl; and Y = Cl and H. Chemical shifts and X-ray data are also reported for o-CH 3 and o-OCH 3 tetrakis(phenoxy)-((XC 6 H 4 O) 4 Ge) and thiophenoxygermanes ((XC 6 H 4 S) 4 Ge). For tetrakis derivatives, 73 Ge resonances are observed for all but the o-methoxyphenoxy compound, for which the inability to detect a resonance is attributed to rapid quadrupolar relaxation caused by intramolecular interactions of the methoxy oxygen with the central atom. The observation of a relatively broad, slightly upfield 73 Ge resonance in the analogous phenyl and thiophenoxy derivatives suggests, as do the results of molecular modeling, that in these compounds there is some hypercoordination. The solid-state structures show bond angles at the aromatic carbon bearing the alkoxy group that suggest an interaction of the alkoxy oxygen with germanium. Oxygen-germanium bond distances are about 17% shorter than the sum of the van der Waals radii.
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