Chasing Protons: How Isothermal Titration Calorimetry, Mutagenesis, and pK_a Calculations Trace the Locus of Charge in Ligand Binding to a tRNA-Binding Enzyme

Abstract

Drug molecules should remain uncharged while traveling through the body and crossing membranes and should only adopt charged state upon protein binding, particularly if charge-assisted interactions can be established in deeply buried binding pockets. Such strategy requires careful pKa design and methods to elucidate whether and where protonation-state changes occur. We investigated the protonation inventory in a series of lin-benzoguanines binding to tRNA-guanine transglycosylase, showing pronounced buffer dependency during ITC measurements. Chemical modifications of the parent scaffold along with ITC measurements, pKa calculations, and site-directed mutagenesis allow elucidating the protonation site. The parent scaffold exhibits two guanidine-type portions, both likely candidates for proton uptake. Even mutually compensating effects resulting from proton release of the protein and simultaneous uptake by the ligand can be excluded. Two adjacent aspartates induce a strong pKa shift at the ligand site, resulting in protonation-state transition. Furthermore, an array of two parallel H-bonds avoiding secondary repulsive effects contributes to the high-affinity binding of the lin-benzoguanines.

Beyond Affinity: Enthalpy–Entropy Factorization Unravels Complexity of a Flat Structure–Activity Relationship for Inhibition of a tRNA-Modifying Enzyme

“…The saltbridge type hydrogen bonds to Asp156 formed by the linbenzoguanines are replaced by weaker charge-assisted contacts in the lin-benzohypoxanthines. 9 Overall, in both ligand series, the parent lin-benzopurine scaffold is completely buried in the protein binding pocket and thus shielded from solvent access.…”

“…The interaction pattern stabilizes the backbone flip and makes a dual ladder of parallel hydrogen bonds to the bound ligand possible, avoiding unfavorable secondary repulsive interactions among the closely approaching hydrogens in the hydrogen-bonding arrays. 9,19,20 Detailed analyses of the protonation inventory overlaid to the binding event showed that N(5) of the lin-benzoguanine scaffold becomes protonated, whereas N(3) remains in the neutral, uncharged state. 9 The lin-benzohypoxanthine scaffold adopts a very similar binding mode, and the interaction pattern to Asp156, Gln203, Gly230, Leu231, and Ala232 is analogously established.…”

“…9,19,20 Detailed analyses of the protonation inventory overlaid to the binding event showed that N(5) of the lin-benzoguanine scaffold becomes protonated, whereas N(3) remains in the neutral, uncharged state. 9 The lin-benzohypoxanthine scaffold adopts a very similar binding mode, and the interaction pattern to Asp156, Gln203, Gly230, Leu231, and Ala232 is analogously established. 8 The lin-benzohypoxanthines lack the exocyclic NH 2 group at the pyrimidinone moiety.…”

“…ITC measurements revealed that the lin-benzohypoxanthines bind to the protein without entrapping a proton at N(5). 9 As a result, the lin-benzohypoxanthines bind significantly more weakly than the lin-benzoguanines. The saltbridge type hydrogen bonds to Asp156 formed by the linbenzoguanines are replaced by weaker charge-assisted contacts in the lin-benzohypoxanthines.…”

“…8 The first modification involves attachment of an exocyclic amino group to a pyrimidinone headgroup, which makes the considered ligands competent to establish enhanced charge-assisted or even saltbridge type hydrogen bonds. 9 The second modification concerns attachment of a series of substituted amino groups at the remote end of the parent scaffold. 10 The binding affinity determined via the dissociation constant (K d value) measured by isothermal titration calorimetry varies only slightly among individual derivatives of the two series and differs among corresponding members of both series by about 1 order of magnitude depending whether the amino group is present or absent at the pyrimidinone moiety.…”