Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway

Abstract

The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP+). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP+ and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants.

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

“…A study with lysozyme showed that an intermediate "unlocked" state is populated to facilitate product release and large-scale conformational fluctuations exist along the catalytic cycle modulating the interaction with the product necessary for releasing it (15). Further, a recent study revealed that dihydrofolate reductase from Escherichia coli releases product in two parallel pathways, an intrinsic pathway and an allosteric pathway, and the enzyme's conformational dynamics plays a critical role in controlling product release (16). Experimentally observed continual change in the coupled protein-water hydrogen-bond dynamics during catalytic cycle further supports our finding that different conformational intermediates are formed during a catalytic cycle (10, 43).…”

“…For example, by what exact mechanism a product exits the active site and the energetically preferential configurations of an enzyme to release the product are still not clear. Few studies have tried to address these questions and related effects on other macroscopic parameters directly through experimental approaches (9,(15)(16)(17)(18)(19). We recently reported that horseradish peroxidase (HRP) can involve multiple intermediate conformational states and pathways during product releasing, and a significant pathway is that the product molecules are spilled out from the enzymatic reaction site; however, releasing the product molecules through a loosely bound enzyme-product state and an open active site remain parallel product releasing pathways (9).…”

Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu

“…A benchmark validation of this method was done using point mutants of known structure. The benchmark consists of nine high-resolution structures of T4 lysozyme point mutants, 29 each differing from the wild type at a single position, and nine variants of Escherichia coli dihydrofolate reductase, 30 each with one or two sequence changes. All lysozyme mutations were V to T or A to S, adding hydroxyl groups.…”

Multi-probe relaxation dispersion measurements increase sensitivity to protein dynamics

“…12, 13 Unfortunately, for DHFR complexes, the sign of Δω can be determined only for a small number of residues, especially in the case of Δω H . 14 Despite this we demonstrate that detailed structural insight can be gained from the absolute chemical shift changes for multiple nuclei when analyzed on the basis of knowledge of the structure of the ground state. Classical analysis of chemical shifts using chemical shift hypersurfaces to relate chemical shifts deviations from random coil values to variations in structural parameters, like backbone torsion angles, hydrogen bonding, ring currents and side-chain torsions gives insight into the structural changes taking place.…”

“…17 All relaxation dispersion experiments were recorded at 301 K and spectrometer frequencies of 500 MHz and 800 MHz. The 15 N and 1 H CPMG relaxation dispersion profiles were recorded using pulse sequences appropriate for protonated samples in an interleaved fashion as previously described by Oyen et al 14 using Poisson gap nonuniform sampling in the indirect dimension. 18 Briefly, a series of scan interleaved CPMG experiments were recorded as a pseudo-3D experiment with 60 % NUS sampling of the full complex data matrices of size 1024 x 128 with 16 scans per FID and a recycle delay of 3.0 seconds.…”