Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase

Thymidylate synthase (TSase) is responsible for synthesizing the sole <i>de novo</i> source of dTMP in all organisms. TSase is a drug target, and as such, it has been well studied in terms of both structure and reaction mechanism. Cysteine 146 in <i>Escherichia coli</i> TSase is universally conserved because it serves as the nucleophile in the enzyme mechanism. Here we use the C146S mutation to probe the role of the sulfur atom in early events in the catalytic cycle beyond serving as the nucleophile. Surprisingly, the single-atom substitution severely decreases substrate binding affinity, and the unfavorable ΔΔ<i>G</i>°_bind is comprised of roughly equal enthalpic and entropic components at 25 °C. Chemical shifts in the free and dUMP-bound states show the mutation causes perturbations throughout TSase, including regions important for complex stability, in agreement with a less favorable enthalpy change. We measured the nuclear magnetic resonance methyl symmetry axis order parameter (<i>S</i>²_axis), a proxy for conformational entropy, for TSase at all vertices of the dUMP binding/C146S mutation thermodynamic cycle and found that the calculated <i>T</i>ΔΔ<i>S</i>°_conf is similar in sign and magnitude to the calorimetric <i>T</i>ΔΔ<i>S</i>°. Further, we ascribed minor resonances in wild-type–dUMP spectra to a state with a covalent bond between Sγ of C146 and C6 of dUMP and find <i>S</i>²_axis values are unaffected by covalent bond formation, indicating this reaction step is neutral with respect to Δ<i>S</i>°_conf. Lastly, the C146S mutation allowed us to measure cofactor analog binding by isothermal titration calorimetry without the confounding heat signature of covalent bond formation. Raltitrexed binds free and singly bound TSase with similar affinities, yet the two binding events have different enthalpy changes, providing further evidence of communication between the two active sites

G121V dependent changes in backbone and side-chain dynamics on the ps-ns timescale.

<p>Changes in backbone <i>S</i>² (A) and τ_e (B) order parameters determined from ¹⁵N relaxation experiments. Changes in side-chain <i>S</i>²_axis (D) and τ_e,_axis (E) order parameters determined from ²H relaxation experiments. Plotted values were obtained by subtracting mutant (E^G121V:NADPH:MTX) from wild-type (E:NADPH:MTX) parameters with significant changes (>1.5σ) highlighted in red (backbone) and blue (side-chain). Significant changes are also mapped onto the structure using red spheres for backbone (C) and blue spheres for side-chain (F). Greens spheres denote residues that have significant changes in both backbone and side-chain order parameters. An area of 5 Å around the active site is highlighted in green.</p

DHFR subdomain and loop nomenclature.

<p>Structure of DHFR complexed with NADPH (purple) and methotrexate (yellow) (pdbid 1RX3). Adenosine binding domain is shown in light blue while the loops domain is shown in wheat. The Met20 (red), F–G (blue), G–H (green) loops are labeled. The site of mutation (G121V) is indicated by a cyan sphere.</p

Model-free analysis of tryptophan indole ¹⁵N relaxation within E:NADPH:MTX and E^G121V:NADPH:MTX complexes.

a<p><i>R</i>_ex values are based on model-free fits.</p

Methotrexate binding traps G121V in the closed conformation.

<p>(A) ¹H–¹⁵N HSQC spectra of E:NADPH:MTX (black) overlain with the ¹H–¹⁵N HSQC spectra of E^G121V:NADPH:MTX (red). (B) The reduced change in chemical shift is calculated using the following formula and is subsequently plotted as a function of residue number: . The changes as a result of mutation (G121V vs. WT) are shown in black and a bona fide closed–occluded change are plotted in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033252#pone.0033252-Osborne1" target="_blank">[23]</a>. The plot shows that the pattern of changes as a result of mutation can be attributed to differences in the local chemical environment and not large-scale structural change. The Met20 (residues 9–23), F–G (residues 117–131), and G–H (residues 142–149) loops are highlighted.</p

Native State Volume Fluctuations in Proteins as a Mechanism for Dynamic Allostery

Allostery enables tight regulation of protein function in the cellular environment. Although existing models of allostery are firmly rooted in the current structure–function paradigm, the mechanistic basis for allostery in the absence of structural change remains unclear. In this study, we show that a typical globular protein is able to undergo significant changes in volume under native conditions while exhibiting no additional changes in protein structure. These native state volume fluctuations were found to correlate with changes in internal motions that were previously recognized as a source of allosteric entropy. This finding offers a novel mechanistic basis for allostery in the absence of canonical structural change. The unexpected observation that function can be derived from expanded, low density protein states has broad implications for our understanding of allostery and suggests that the general concept of the native state be expanded to allow for more variable physical dimensions with looser packing

Protein Mass Effects on Formate Dehydrogenase

Isotopically labeled enzymes (denoted as “heavy” or “Born–Oppenheimer” enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein’s higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein’s mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born–Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born–Oppenheimer enzyme hypothesis (vibrational) and also slower time scale events that are non-Born–Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor–acceptor distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry and the protein’s macromolecular electrostatics at slower time scales; that is, both Born–Oppenheimer and non-Born–Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however, making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins

Mg²⁺ Binds to the Surface of Thymidylate Synthase and Affects Hydride Transfer at the Interior Active Site

Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e., the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg²⁺ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg²⁺ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg²⁺ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg²⁺ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg²⁺ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg²⁺ accelerates the hydride transfer by ∼7-fold but does not affect the magnitude or temperature dependence of the intrinsic kinetic isotope effect. These results suggest that Mg²⁺ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg²⁺ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg²⁺ with the glutamyl tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with polyglutamyl substitutes as species-specific antibiotic drugs