Effects of the Donor–Acceptor Distance and Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed Reaction

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor–acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD’s rearrangement toward tunneling-ready states. This structure–function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C–H→C transfer reactions

Comparison of the best poses found with Glide/XP and GOLD for selected drugs binding to open hERG1 channel.

<p>In parentheses is the population of the cluster from which the best pose comes from (always most populated one). In the case of Glide/XP, the output is only giving the selected best poses using a selection criteria explained in the methods. For definitions of the binding pockets see Durdagi et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105553#pone.0105553-Durdagi1" target="_blank">[7]</a>.</p><p>Comparison of the best poses found with Glide/XP and GOLD for selected drugs binding to open hERG1 channel.</p

Substitutions in benzene ring (position R3) are determinants of prolongation of deactivation.

<p>Electrophysiologic responses to four molecules, MC-II-163c, MC-II-159C, MC-II-157b, and MC-II-43c are compared. Panel A shows the structures. All four molecules are similar except for the substituents on benzene ring #2. MC-II-43c is unsubstituted, whereas MC-II-163c, MC-II-159C, MC-II-157b are <i>para</i> substituted with fluoro, bromo and tri-fluoro groups respectively. Panel B shows raw examples of the slowing in the deactivation rate. Panel C shows the deactivation time constants relative to the base lines. Panel D shows the magnitude of the tail current relative to baseline. Panel E shows the shift in the voltage-dependence of activation and Panel F shows the shift in the voltage-dependent of inactivation. Panels C-F show no significant differences between the molecules except for their effects on deactivation. All 4 molecules shifted voltage dependence of activation to hyperpolarized potentials.</p

Schematic representation of the studied compounds topology showing the different R1, R2 and R3.

<p>The groups were identified to be critical determinants of high-affinity/high-specificity binding of activator to site located in S4–S5 linker of the hERG1 channel. Atom N* depicted in blue represents tentative protonation site. The black arrow represents the versor (∧b) perpendicular to the plane defined by atoms N, C, O, N, C and O of the polyamide moiety, common structure element present in all molecules structure. Top panel shows NS-1643 and chemical group identification. Bottom panel illustrates compound groups synthesized.</p

The concentration-response relationship of MC-II-157c.

<p>Panel A shows the raw currents elicited by the protocol shown in the inset. Panels B-E show the concentration-response relationships for mean tail current amplitude (Panel B), mean Δ shift in V_1/2 of activation (Panel C), mean Δ shift in V_1/2 of inactivation in Panel D and mean prolongation of the deactivation in Panel E.</p

Concentration dependence of compounds MC-I-159b, MC-I-169b and MC-I-155b.

<p>Panel A: Chemical structures of compounds are shown. Panel B: Time dependent changes of tail current magnitudes in response to different drug concentrations. Panel C: The magnitude of the tail current relative to baseline. Panel D: The shifts in the voltage-dependence of activation and in the voltage-dependent of inactivation. The deactivation time constants relative to base lines is represented at bottom-right Panel E.</p

Synthesis of NS1643 Analogs and Intermediates (Scheme 1).

<p>Synthesis of NS1643 Analogs and Intermediates (Scheme 1).</p

Pharmacologic response (Δ) to NS1643 (open white bars at 10 µM), MC-II-157c (black bars at 10 µM) and MC-II-159C (grey bars at 10 µM) are compared in wild type (WT) versus E544L.

<p>Panel A: In WT, NS1643 MC-II-157c and MC-II-159c, all shift voltage-dependence of activation and Panel B: slow deactivation. (top) In E544L, pharmacologic response to NS1643 is exaggerated whereas Δ response to MC-II-157c and Δ MC-II-159c were markedly diminished. (bottom-left) Panel C: In terms of amplitude of the tail current, in E544L response to NS1643 is exaggerated whereas response to MC-II-157c and MC-II-159C is markedly diminished. Pharmacologic response in terms of inactivation is complex. Panel D: In WT, MC-II-157c shifts voltage-dependence of inactivation to depolarized potentials whereas MC-II-159C shifts voltage dependence to hyperpolarized potentials. Pharmacologic response to NS1643 is exaggerated in E544L whereas for MC-II-157c and MC-II-159C responses are diminished. (bottom-right) * evaluates the statistical significance of the Δ response to NS1643 compared to Δ response to MC-II-157c or Δ response MC-II-159c. * designates p<0.05; ** designates p<0.01. n values were: For Activation panel in WT n = 10,8 and 3; for E544L n = 9,6 and 3. For deactivation in WT n = 8,8,3 respectively and for E544L n = 4,6,3. For tail current amplitude, in WT n = 9,8,3 and in E544L n = 8,6,2. For inactivation, n = 9,8,3 and for E544L n = 5,6,2.</p

Mapping of the bound conformations for MC-II-157c at the S4–S5 domain of the receptor.

<p>2D ligand interactions diagram (left-bottom panel) and surface representation of docked pose (right-bottom panel) are also shown at the figure.</p

DataSheet1.PDF

<p>I_Kr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of I_Kr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac I_kr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.</p