Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase

The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-[superscript 2]H], [2′-[superscript 2]H], [4′-[superscript 2]H], [5′-[superscript 2]H], [U−[superscript 13]C, [superscript 15]N], [U−[superscript 15]N], and [5,6 -[superscript 2]H] CDP and [β-[superscript 2]H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of [superscript 2]H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.National Institutes of Health (U.S.) (GM29595)(EB002804)(EB002026

Quantifying Water-Mediated Protein–Ligand Interactions in a Glutamate Receptor: A DFT Study

It is becoming increasingly clear that careful treatment of water molecules in ligand–protein interactions is required in many cases if the correct binding pose is to be identified in molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors. Despite possessing similar chemical moieties, crystal structures of glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in complex with the ligand-binding core of the GluA2 ionotropic glutamate receptor revealed, contrary to all expectation, two distinct modes of binding. The difference appears to be related to the position of water molecules within the binding pocket. However, it is unclear exactly what governs the preference for water molecules to occupy a particular site in any one binding mode. In this work we use density functional theory (DFT) calculations to investigate the interaction energies and polarization effects of the various components of the binding pocket. Our results show (i) the energetics of a key water molecule are more favorable for the site found in the glutamate-bound mode compared to the alternative site observed in the AMPA-bound mode, (ii) polarization effects are important for glutamate but less so for AMPA, (iii) ligand–system interaction energies alone can predict the correct binding mode for glutamate, but for AMPA alternative modes of binding have similar interaction energies, and (iv) the internal energy is a significant factor for AMPA but not for glutamate. We discuss the results within the broader context of rational drug-design