How Nothing Boosts Affinity: Hydrophobic Ligand Binding to the Virtually Vacated S₁′ Pocket of Thermolysin

We investigated the hydration state of the deep, well-accessible hydrophobic S₁′ specificity pocket of the metalloprotease thermolysin with purposefully designed ligands using high-resolution crystallography and isothermal titration calorimetry. The S₁′ pocket is known to recognize selectively a very stringent set of aliphatic side chains such as valine, leucine, and isoleucine of putative substrates. We engineered a weak-binding ligand covering the active site of the protease without addressing the S₁′ pocket, thus transforming it into an enclosed cavity. Its sustained accessibility could be proved by accommodating noble gas atoms into the pocket in the crystalline state. The topology and electron content of the enclosed pocket with a volume of 141 ų were analyzed using an experimental MAD-phased electron density map that was calibrated to an absolute electron number scale, enabling access to the total electron content within the cavity. Our analysis indicates that the S₁′ pocket is virtually vacated, thus free of any water molecules. The thermodynamic signature of the reduction of the void within the pocket by growing aliphatic P₁′ substituents (H, Me, <i>i</i>Pr, <i>i</i>Bu) reveals a dramatic, enthalpy-dominated gain in free energy of binding resulting in a factor of 41 000 in <i>K</i>_d for the H-to-<i>i</i>Bu transformation. Substituents placing polar decoy groups into the pocket to capture putatively present water molecules could not collect any evidence for a bound solvent molecule

Ligand Binding Stepwise Disrupts Water Network in Thrombin: Enthalpic and Entropic Changes Reveal Classical Hydrophobic Effect

Well-ordered water molecules are displaced from thrombin’s hydrophobic S3/4-pocket by P3-varied ligands (Gly, d-Ala, d-Val, d-Leu to d-Cha with increased hydrophobicity and steric requirement). Two series with 2-(aminomethyl)-5-chlorobenzylamide and 4-amidinobenzylamide at P1 were examined by ITC and crystallography. Although experiencing different interactions in S1, they display almost equal potency. For both scaffolds the terminal benzylsulfonyl substituent differs in binding, whereas the increasingly bulky P3-groups address S3/4 pocket similarly. Small substituents leave the solvation pattern unperturbed as found in the uncomplexed enzyme while increasingly larger ones stepwise displace the waters. Medium-sized groups show patterns with partially occupied waters. The overall 40-fold affinity enhancement correlates with water displacement and growing number of van der Waals contacts and is mainly attributed to favorable entropy. Both Gly derivatives deviate from the series and adopt different binding modes. Nonetheless, their thermodynamic signatures are virtually identical with the homologous d-Ala derivatives. Accordingly, unchanged thermodynamic profiles are no reliable indicator for conserved binding modes

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 <i>lin</i>-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

Chasing Protons: How Isothermal Titration Calorimetry, Mutagenesis, and p<i>K</i>_a 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 p<i>K</i>_a design and methods to elucidate whether and where protonation-state changes occur. We investigated the protonation inventory in a series of <i>lin</i>-benzoguanines binding to tRNA−guanine transglycosylase, showing pronounced buffer dependency during ITC measurements. Chemical modifications of the parent scaffold along with ITC measurements, p<i>K</i>_a 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 p<i>K</i>_a 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 <i>lin</i>-benzoguanines

New Insights into Human 17β-Hydroxysteroid Dehydrogenase Type 14: First Crystal Structures in Complex with a Steroidal Ligand and with a Potent Nonsteroidal Inhibitor

17β-HSD14 is a SDR enzyme able to oxidize estradiol and 5-androstenediol using NAD⁺. We determined the crystal structure of this human enzyme as the holo form and as ternary complexes with estrone and with the first potent, nonsteroidal inhibitor. The structures reveal a conical, rather large and lipophilic binding site and are the starting point for structure-based inhibitor design. The two natural variants (S205 and T205) were characterized and adopt a similar structure

Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.

<p>Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.</p

Substrate base binding pocket of <i>Z.</i> <i>mobilis</i> Tgt and modelled human Tgt.<b> </b>

<p>A) Detail of <i>Z. mobilis</i> Tgt·preQ₁ complex crystal structure (PDB-code: <b><u>1p0e</u></b>) showing the active site with the bound substrate in stick representation. Carbon atoms of protein residues are coloured in green, those of preQ₁ in orange. B) Homology model of human Tgt created with the <i>Z. mobilis</i> Tgt crystal structure as a template. The close up shows active site residues (carbon atoms in grey) superimposed with preQ₁ (carbon atoms in orange) as present in <b><u>1p0e</u></b>. The coordinates of the homology model are provided within the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s001" target="_blank">Coordinates S1</a>).</p

Crystal structures of Tgt variants in complex with preQ₁ or queuine: surface representation of substrate pockets.

<p>The solvent accessible surfaces of active sites from <i>Z. mobilis</i> Tgt variants are shown in bright yellow. The respective Tgt variant plus the bound ligand (shown in stick representation) are indicated in each sub-figure. Also amino acid residues at positions 158 and 233 are shown in stick representation. Carbon atoms of original amino acids and of the bound ligand are coloured green, those of mutated amino acids magenta. As the electron density assignable to the dihydroxy-cyclopentenyl moiety of queuine is poorly defined in all structures containing this ligand the coordinates of this moiety are not present in the respective structures deposited with the Protein Data Base. Accordingly, the conformations of the dihydroxy-cyclopentenyl shown in (D), (F) and (H) are tentative. To indicate this fact, the carbon atoms of this moiety are shown in grey. Selected water molecules are shown as red spheres. 2|F_o|-|F_c| (at σ 1.0) electron density is shown for the bound ligand and water molecules. 2|F_o|-|F_c| electron density contoured at a σ level of 1.0 is coloured blue, |F_o|-|F_c| electron density contoured at a σ level of 2.5 is coloured magenta. An overview of the crystal structures analysed in this study including resolutions and PDB codes is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s004" target="_blank">Table S1</a>.</p

Kinetic parameters for “wild type” Tgt and mutated Tgt variants.^*

<p>Kinetic parameters for “wild type” Tgt and mutated Tgt variants.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#nt101" target="_blank">*</a>}</p

Crystal structures of Tgt variants in complex with preQ₁ or queuine: stick representation of substrate pockets.

<p>The respective Tgt variant plus the bound ligand are indicated in each sub-figure. Carbon atoms of original amino acids are coloured green, those of mutated amino acids as well as of the bound ligand orange. As the electron density assignable to the dihydroxy-cyclopentenyl moiety of queuine is poorly defined in all structures containing this ligand the coordinates of this moiety are not present in the respective structures deposited with the Protein Data Base. Accordingly, the conformations of the dihydroxy-cyclopentenyl shown in (D), (F) and (H) are tentative. To indicate this fact, the carbon atoms of this moiety are shown in grey. Selected water molecules are shown as red spheres. Electron density is shown for amino acid residues at positions 158 and 233 as well as for the ligand and for water molecules. The 2|F_o|-|F_c| electron density map contoured at a σ level of 1.0 is coloured blue. The green density represents an |F_o|-|F_c| omit map contoured at 2.5 σ. An overview of the crystal structures analysed in this study including nominal resolutions and PDB codes is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s004" target="_blank">Table S1</a>.</p