Occupying a flat subpocket in a tRNA-modifying enzyme with ordered or disordered side chains: Favorable or unfavorable for binding?

Small-molecule ligands binding with partial disorder or enhanced residual mobility are usually assumed as unfavorable with respect to their binding properties. Considering thermodynamics, disorder or residual mobility is entropically favorable and contributes to the Gibbs energy of binding. In the present study, we analyzed a series of congeneric ligands inhibiting the tRNA-modifying enzyme TGT. Attached to the parent lin-benzoguanine scaffold, substituents in position 2 accommodate in a flat solvent-exposed pocket and exhibit varying degree of residual mobility. This is indicated in the crystal structures by enhanced B-factors, reduced occupancies, or distributions over split conformers. MD simulations of the complexes suggest an even larger scatter over several conformational families. Introduction of a terminal acidic group fixes the substituent by a salt-bridge to an Arg residue. Overall, all substituted derivatives show the same affinity underpinning that neither order nor disorder is a determinant factor for binding affinity. The additional salt bridge remains strongly solvent-exposed and thus does not contribute to affinity. MD suggests temporary fluctuation of this contact.

Chasing protons: how isothermal titration calorimetry, mutagenesis, and pKa calculations trace the locus of charge in ligand binding to a tRNA-binding enzyme.

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.

Lead optimization focuses on binding-affinity improvement. If a flat structure-activity relationship is detected, usually optimization strategies are abolished as unattractive. Nonetheless, as affinity is composed of an enthalpic and entropic contribution, factorization of both can unravel the complexity of a flat, on first sight tedious SAR. In such cases, the binding free energy of different ligands can be rather similar, but it can factorize into enthalpy and entropy distinctly. We investigated the thermodynamic signature of two classes of lin-benzopurines binding to tRNA-guanine transglycosylase. While the differences are hardly visible in the free energy, they involve striking enthalpic and entropic changes. Analyzing thermodynamics along with structural features revealed that one ligand set binds to the protein without inducing significant changes compared to the apo structure; however, the second series provokes complex adaptation, leading to a conformation similar to the substrate-bound state. In the latter state, a cross-talk between two pockets is suggested.

Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics.

BACKGROUND: The thermodynamic characterization of protein-ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations. METHODS: Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry. RESULTS AND CONCLUSIONS: We used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4kJ⋅mol(-1), which is considered significant. GENERAL SIGNIFICANCE: Our results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein-ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature.

Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals a novel active-site environment.

Coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum (Linaceae) catalyzes the unusual methylation of the side-chain hydroxyl group of coniferyl alcohol. The protein was heterologously expressed in Escherichia coli as a hexahistidine derivative and purified for crystallization. Diffracting crystals were obtained of the pure protein and of its selenomethionine derivative, as well as of complexes with coniferyl alcohol and with S-adenosyl-L-homocysteine together with coniferyl alcohol 9-O-methyl ether (PDB entries 4ems, 4e70 and 4evi, respectively). The X-ray structures show that the phenylpropanoid binding mode differs from other phenylpropanoid O-methyltransferases such as caffeic acid O-methyltransferase. Moreover, the active site lacks the usually conserved and catalytic histidine residue and thus implies a different reaction mode for methylation. Site-directed mutagenesis was carried out to identify critical amino acids. The binding order of coniferyl alcohol and S-adenosyl-L-methionine was investigated by isothermal titration calorimetry experiments.

Targeting the blind spot of polycationic nanocarrier-based siRNA delivery.

Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA vs PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA vs pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation-siRNA and polycation-pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems.