Effect of Halogen Substitutions on dUMP to Stability of Thymidylate Synthase/dUMP/mTHF Ternary Complex Using Molecular Dynamics Simulation

The stability of the thymidylate synthase (TS)/2-deoxyuridine-5-monophosphate (dUMP)/5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) ternary complex formation and Michael addition are considered as important steps that are involved in the inhibition mechanism of the anticancer prodrug 5-fluorouracil (5-FU). Here, the effect of three different halogen substitutions on the C-5 position of the dUMP (XdUMPs = FdUMP, CldUMP, and BrdUMP), the normal substrate, on the stability of the TS/dUMP and TS/dUMP/mTHF binary and ternary complexes, respectively, was investigated via molecular dynamics simulation. The simulated results revealed that the stability of all the systems was substantially increased by mTHF binding to the catalytic pocket. In the ternary complex, a much greater stabilization of the dUMP and XdUMPs through electrostatic interactions, including charge–charge and hydrogen bond interactions, was found compared to mTHF. An additional unique hydrogen bond between the substituted fluorine of FdUMP and the hydroxyl group of the TS Y94 residue was observed in both the binary and ternary complexes. The distance between the S^– atom of the TS C146 residue and the C6 atom of dUMP, at <4 Å in all systems, suggested that a Michael addition with the formation of a S–C6 covalent bond potentially occurred, although the hydrogen atom on C6 of dUMP is substituted by a halogen atom. The MM/PBSA binding free energy revealed the significant role of the bridging waters around the ligands in the increased binding affinity (∼10 kcal/mol) of dUMP/XdUMP, either alone or together with mTHF, toward TS. The order of the averaged binding affinity in the ternary systems was found to be CldUMP ≈ FdUMP > dUMP > BrdUMP, suggesting that CldUMP could be a potent candidate TS inhibitor, the same as FdUMP (the metabolite form of 5-FU)

High-Level QM/MM Calculations Support the Concerted Mechanism for Michael Addition and Covalent Complex Formation in Thymidylate Synthase

Thymidylate synthase (TS) is a promising cancer target, due to its crucial function in thymine synthesis. It performs the reductive methylation of 2′-deoxyuridine-5′-phosphate (dUMP) to thymidine-5′-phosphate (dTMP), using <i>N</i>-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent conformational activation, this complex has been proposed to react via nucleophilic attack (Michael addition) by Cys146, followed by methylene-bridge formation to generate the ternary covalent intermediate. Herein, QM/MM (B3LYP-D/6-31+G­(d)-CHARMM27) methods are used to model the formation of the ternary covalent intermediate. A two-dimensional potential energy surface reveals that the methylene-bridged intermediate is formed via a concerted mechanism, as indicated by a single transition state on the minimum energy pathway and the absence of a stable enolate intermediate. A range of different QM methods (B3LYP, MP2 and SCS-MP2, and different basis sets) are tested for the calculation of the activation energy barrier for the formation of the methylene-bridged intermediate. We test convergence of the QM/MM results with respect to size of the QM region. Inclusion of Arg166, which interacts with the nucleophilic thiolate, in the QM region is important for reliable results; the MM model apparently does not reproduce energies for distortion of the guanidinium side chain correctly. The spin component scaled-Møller–Plessett perturbation theory (SCS-MP2) approach was shown to be in best agreement (within 1.1 kcal/mol) while the results obtained with MP2 and B3LYP also yielded acceptable values (deviating by less than 3 kcal/mol) compared with the barrier derived from experiment. Our results indicate that using a dispersion-corrected DFT method, or a QM method with an accurate treatment of electron correlation, increases the agreement between the calculated and experimental activation energy barriers, compared with the semiempirical AM1 method. These calculations provide important insight into the reaction mechanism of TS and may be useful in the design of new TS inhibitors

Molecular Dynamics Simulation Reveals the Selective Binding of Human Leukocyte Antigen Alleles Associated with Behçet's Disease

<div><p>Behçet’s disease (BD), a multi-organ inflammatory disorder, is associated with the presence of the human leukocyte antigen (HLA) HLA-B*51 allele in many ethnic groups. The possible antigen involvement of the major histocompatibility complex class I chain related gene A transmembrane (MICA-TM) nonapeptide (AAAAAIFVI) has been reported in BD symptomatic patients. This peptide has also been detected in HLA-A*26:01 positive patients. To investigate the link of BD with these two specific HLA alleles, molecular dynamics (MD) simulations were applied on the MICA-TM nonapeptide binding to the two BD-associated HLA alleles in comparison with the two non-BD-associated HLA alleles (B*35:01 and A*11:01). The MD simulations were applied on the four HLA/MICA-TM peptide complexes in aqueous solution. As a result, stabilization for the incoming MICA-TM was found to be predominantly contributed from van der Waals interactions. The P2/P3 residue close to the N-terminal and the P9 residue at the C-terminal of the MICA-TM nonapeptide served as the anchor for the peptide accommodated at the binding groove of the BD associated HLAs. The MM/PBSA free energy calculation predicted a stronger binding of the HLA/peptide complexes for the BD-associated HLA alleles than for the non-BD-associated ones, with a ranked binding strength of B*51:01 > B*35:01 and A*26:01 > A*11:01. Thus, the HLAs associated with BD pathogenesis expose the binding efficiency with the MICA-TM nonapeptide tighter than the non-associated HLA alleles. In addition, the residues 70, 73, 99, 146, 147 and 159 of the two BD-associated HLAs provided the conserved interaction for the MICA-TM peptide binding.</p></div

Structural basis of HLA class I.

<p>(A) Schematic model of HLA buried in the transmembrane. (B) HLA (pink) contains the <i>α</i>1 and <i>α</i>2 subdomains that contribute to the peptide binding groove, while <i>α</i>3 is the C-terminal domain in complex with <i>ß</i>₂-microgluobulin (<i>ß</i>₂m) as a noncovalently supported protein (cyan). (C) Ribbon and (D) van der Waals surface representations of the MICA-TM nonapeptide (green stick model) occupied in the peptide binding sub-sites (S1–S9, shaded by different colors) of HLA-B*51:01.</p

Hydrogen bond interactions.

<p>The percentage occupancy of H-bonds averaged over the last 25 ns of simulation time between the nine residues (P1–P9) of the MICA-TM peptide and the HLA residues for the four complexes.</p

The binding free energy and energy components (kcal/mol) for the four HLA/MICA-TM complexes predicted by the MM/PBSA method.

<p>HLA alleles are</p><p>^<i>a</i> associated or</p><p>^<i>b</i> not associated with Behçet’s disease (BD).</p><p>Data are shown as the mean ± SD, derived from independent simulations. Means within a paired row (HLA-A or HLA-B alleles that are associated with BD versus that are not) followed by a different letter are significantly different.</p><p>Δ<i>G</i>_<i>bind</i> is the binding energy with inclusion of entropic term.</p><p>The binding free energy and energy components (kcal/mol) for the four HLA/MICA-TM complexes predicted by the MM/PBSA method.</p

Structural flexibilities of the HLA alleles bound with the MICA-TM peptide.

<p>Structural flexibilities were evaluated by B-factor. The ribbon color changes from blue (rigid) to red (flexible) to represent a low to high protein flexibility. Note that for clarity only the binding groove structure and the MICA-TM peptide are shown.</p

Decomposition energy per HLA residue fingerprint plots.

<p>The HLA contribution to the MICA-TM binding is shown in terms of the electrostatic (ele) and van der Waals (vdW) interactions.</p

Averaged decomposition energy contributions in HLA binding to MICA-TM.

<p>Per-residue decomposition energies and the energy components in terms of the electrostatic (ele) and van der Waals (vdW) interactions for the P1–P9 residues of MICA-TM.</p