Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the <i>p</i>-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on <i>k</i>_cat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)­propyl]­carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the <i>p</i>-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The <i>K</i>_i for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N‑Termini

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of <i>Escherichia coli</i> DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (<i>R</i>_g) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the <i>R</i>_g increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated

Aspects of Weak Interactions between Folate and Glycine Betaine

Folate, or vitamin B₉, is an important compound in one-carbon metabolism. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equilibrium toward the free ligand and protein species. This study uses vapor-pressure osmometry to explore the interaction of folate with the model osmolyte, glycine betaine. This method yields a preferential interaction potential (μ₂₃/<i>RT</i> value). This value is concentration-dependent as folate dimerizes. The μ₂₃/<i>RT</i> value also tracks the deprotonation of folate’s N3–O4 keto–enol group, yielding a p<i>K</i>_a of 8.1. To determine which folate atoms interact most strongly with betaine, the interaction of heterocyclic aromatic compounds (as well as other small molecules) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the μ₂₃/<i>RT</i> values into α values for atom types was achieved. This allows prediction of μ₂₃/<i>RT</i> values for larger molecules such as folate. Molecular dynamics simulations of folate show a variety of structures from extended to L-shaped. These conformers possess μ₂₃/<i>RT</i> values from −0.18 to 0.09 <i>m</i>^–1, where a negative value indicates a preference for solvation by betaine and a positive value indicates a preference for water. This range of values is consistent with values observed in osmometry and solubility experiments. As the average predicted folate μ₂₃/<i>RT</i> value is near zero, this indicates folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water, while the aromatic rings prefer betaine. In general, the more protonated species in our small molecule survey interact better with betaine as they provide a source of hydrogens (betaine is not a hydrogen bond donor). Upon deprotonation of the small molecule, the preference swings toward water interaction because of its hydrogen bond donating capacities

Highly Dynamic Anion–Quadrupole Networks in Proteins

The dynamics of anion–quadrupole (or anion−π) interactions formed between negatively charged (Asp/Glu) and aromatic (Phe) side chains are for the first time computationally characterized in RmlC (Protein Data Bank entry 1EP0), a homodimeric epimerase. Empirical force field-based molecular dynamics simulations predict anion–quadrupole pairs and triplets (anion–anion−π and anion−π–π) are formed by the protein during the simulated trajectory, which suggests that the anion–quadrupole interactions may provide a significant contribution to the overall stability of the protein, with an average of −1.6 kcal/mol per pair. Some anion−π interactions are predicted to form during the trajectory, extending the number of anion–quadrupole interactions beyond those predicted from crystal structure analysis. At the same time, some anion−π pairs observed in the crystal structure exhibit marginal stability. Overall, most anion−π interactions alternate between an “on” state, with significantly stabilizing energies, and an “off” state, with marginal or null stabilizing energies. The way proteins possibly compensate for transient loss of anion–quadrupole interactions is characterized in the RmlC aspartate 84–phenylalanine 112 anion–quadrupole pair observed in the crystal structure. A double-mutant cycle analysis of the thermal stability suggests a possible loss of anion−π interactions compensated by variations of hydration of the residues and formation of compensating electrostatic interactions. These results suggest that near-planar anion–quadrupole pairs can exist, sometimes transiently, which may play a role in maintaining the structural stability and function of the protein, in an otherwise very dynamic interplay of a nonbonded interaction network as well as solvent effects

Modulating Enzyme Activity by Altering Protein Dynamics with Solvent

Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role of enzyme structure is well recognized; however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aqueous mixture of organic solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from <i>Escherichia coli</i> in aqueous mixtures of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (v/v) IPA concentration. Stopped-flow kinetic measurements find that the pH-independent <i>k</i>_hydride rate decreases by 2.2 fold. X-ray crystallographic enzyme structures show no noticeable differences, while computational studies indicate that the transition state and electrostatic effects were identical for water and mixed solvent conditions; quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzymeʼs ability to access its functionally relevant conformational substates, explaining the decreased <i>k</i>_hydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p