Water Makes the Difference: Rearrangement of Water Solvation Layer Triggers Non‐additivity of Functional Group Contributions in Protein–Ligand Binding

Abstract

The binding of four congeneric peptide-like thermolysin inhibitors has been studied by high-resolution crystal structure analysis and isothermal titration calorimetry. The ligands differ only by a terminal carboxylate and/or methyl group. A surprising non-additivity of functional group contributions for the carboxylate and/or methyl groups is detected. Adding the methyl first and then the carboxylate group results in a small Gibbs free energy increase and minor enthalpy/entropy partitioning for the first modification, whereas the second involves a strong affinity increase combined with large enthalpy/entropy changes. However, first adding the carboxylate and then the methyl group yields reverse effects: the acidic group attachment now causes minor effects, whereas the added methyl group provokes large changes. As all crystal structures show virtually identical binding modes, affinity changes are related to rearrangements of the first solvation layer next to the S(2)' pocket. About 20-25 water molecules are visible next to the studied complexes. The added COO(-) groups perturb the local water network in both carboxylated complexes, and the attached methyl groups provide favorable interaction sites for water molecules. Apart from one example, a contiguously connected water network between protein and ligand functional groups is observed in all complexes. In the complex with the carboxylated ligand, which still lacks the terminal methyl group, the water network is unfavorably ruptured. This results in a surprising thermodynamic signature showing only a minor affinity increase upon COO(-) group attachment. Because the further added methyl group provides a favorable interaction site for water, the network can be reestablished, and a strong affinity increase with a large enthalpy/entropy signature is then detected.

The Impact of Binding Thermodynamics on Medicinal Chemistry Optimizations

“…Another factor that affects binding thermodynamics is the rigid body entropy loss that is estimated to be 15-20 kJ/mol [19] and fragments are able to compensate this entropy loss at a lesser extent than larger compounds. It is worth also noting that the perturbation of the water network upon binding although associated with significant contribution to binding thermodynamics [36,37] is less important for fragments than for larger size ligands. Summarizing, fragments have a binding thermodynamic profile characterized by enthalpy domination and this, first, distinguishes them from larger ligands and, second, forms the thermodynamic rationale of fragment based approaches.…”

Is there a link between selectivity and binding thermodynamics profiles?

“…Saquinavir and TMC-126 have the same number of polar groups, however Saquinavir binding is associated with unfavourable ~+5 kJ/mol binding enthalpy while TMC-126 binding is significantly more enthalpic (H~-50 kJ/mol) due to the better orientation of its polar groups [7]. It should also be noted that binding-site water molecules have complex influence on thermodynamics signatures [8][9][10]. Orientation of polar groups largely influences their specific interactions compared to non-polar functional groups that are introduced to fill apolar cavities.…”

“…Replacement of unstable water molecules within hydrophobic pockets is mostly driven by entropy changes, although enthalpy gain coupled with water replacement by apolar moieties had also been reported [8][9]. The effect of binding site waters has been recently reviewed by using WaterMap for solvation energetic calculations [11].…”

Zerlegung des hydrophoben Effekts auf molekularer Ebene: Die Rolle von Wasser, Enthalpie und Entropie bei der Ligandenbindung an Thermolysin

“…Kristallstrukturen mit den Liganden 1, 2 und 7 wurden bereits in früheren Arbeiten bestimmt. [6,30] Daten für TLN-7 wurden neu verfeinert, wobei der besseren Vergleichbarkeit willen dasselbe Protokoll für alle untersuchten Liganden angewendet wurde. Da der Bindungsmodus der Gerüststruktur bereits im Detail beschrieben wurde, [30] werden hier in kurzer Form lediglich die zentralen Aspekte des Bindungsmodus der Gerüststruktur angesprochen.…”

“…Die Kristallstrukturen von TLN-1 und TLN-2 wurden in einer früheren Studie verglichen. [30] Das Glycin-Derivat 1 ist über zwei Wassermoleküle mit der Carbonylgruppe in der Hauptkette von Asn111 verbrückt. Beide Wassermoleküle werden dagegen beim TLN-2-Komplex aufgrund sterischer Konflikte durch die angefügte Methylgruppe im Alanin-Derivat 2 verdrängt (Abbildung 1 a, eingekreist in Hellblau).…”