Hydrogen atoms play a key role in protein-ligand recognition. They determine the quality of established H-bonding networks and define the protonation of bound ligands. Structural visualization of H atoms by X-ray crystallography is rarely possible. We used neutron diffraction to determine the positions of the hydrogen atoms in the ligands aniline and 2-aminopyridine bound to the archetypical serine protease trypsin. The resulting structures show the best resolution so far achieved for proteins larger than 100 residues and allow an accurate description of the protonation states and interactions with nearby water molecules. Despite its low pK of 4.6 and a large distance of 3.6 Å to the charged Asp189 at the bottom of the S1 pocket, the amino group of aniline becomes protonated, whereas in 2-aminopyridine, the pyridine nitrogen picks up the proton although its amino group is 1.6 Å closer to Asp189. Therefore, apart from charge-charge distances, tautomer stability is decisive for the resulting binding poses, an aspect that is pivotal for predicting correct binding.

Structural fixation of a ligand in its bioactive conformation may, due to entropic reasons, improve affinity. We present a congeneric series of thrombin ligands with a variety of functional groups triggering preorganization prior to binding. Fixation in solution and complex formation have been characterized by crystallography, isothermal titration calorimetry (ITC), and molecular dynamics (MD) simulations. First, we show why these preorganizing modifications do not affect the overall binding mode and how key interactions are preserved. Next, we demonstrate how preorganization thermodynamics can be largely dominated by enthalpy rather than entropy because of the significant population of low-energy conformations. Furthermore, a salt bridge is shielded by actively reducing its surface exposure, thus leading to an enhanced enthalpic binding profile. Our results suggest that the consideration of the ligand solution ensemble by MD simulation is necessary to predict preorganizing modifications that enhance the binding behavior of already promising binders.

The transient specificity pocket of aldose reductase only opens in response to specific ligands. This pocket may offer an advantage for the development of novel, more selective ligands for proteins with similar topology that lack such an adaptive pocket. Our aim was to elucidate which properties allow an inhibitor to bind in the specificity pocket. A series of inhibitors that share the same parent scaffold but differ in their attached aromatic substituents were screened using ITC and X-ray crystallography for their ability to occupy the pocket. Additionally, we investigated the electrostatic potentials and charge distribution across the attached terminal aromatic groups with respect to their potential to bind to the transient pocket of the enzyme using ESP calculations. These methods allowed us to confirm the previously established hypothesis that an electron-deficient aromatic group is an important prerequisite for opening and occupying the specificity pocket. We also demonstrated from our crystal structures that a pH shift between 5 and 8 does not affect the binding position of the ligand in the specificity pocket. This allows for a comparison between thermodynamic and crystallographic data collected at different pH values.

In the S1 pocket, the serine proteases thrombin and trypsin commonly feature Asp189 and a Ala190Ser and Glu192Gln exchange. Nevertheless, thrombin cleaves peptide chains solely after Arg, and trypsin after Lys and Arg. Thrombin exhibits a Na+-binding site next to Asp189, which is missing in trypsin. The fragment benzylamine shows direct H-bonding to Asp189 in trypsin, while in thrombin, it forms an H-bond to Glu192. A series of fragments and expanded ligands were studied against both enzymes and mutated variants by crystallography and ITC. The selectivity-determining features of both S1 pockets are difficult to assign to one dominating factor. The Ala190Ser and Glu192Gln replacements may be regarded as highly conserved as no structural and affinity changes are observed between both proteases. With respect to charge distribution, Glu192, together with the thrombin-specific sodium ion, helps in creating an electrostatic gradient across the S1 pocket. This feature is definitely absent in trypsin but important for selectivity along with solvation-pattern differences in the S1 pocket.

Wasserstoffatome sind entscheidend bei der Vermittlung von Protein-Ligand-Wechselwirkungen. Sie bestimmen die Qualitäte ntstehender H-Brückennetzwerke und definieren Protonierungszustände gebundener Liganden. Die strukturelle Visualisierung von H-Atomen ist rçntgenkristallographischn ur selten mçglich.H ier wurden die Wasserstoffpositionen von Anilin und 2-Aminopyridin im an die Serinprotease Trypsin gebundenen Zustand durch Neutronenbeugung untersucht. Die resultierenden Strukturen wurden mit der hçchsten bisher fürProteine mit > 100 Aminosäuren erzielten Auflçsung erhalten. Dies ermçglicht die exakte Beschreibung der Protonierungszustände und Wechselwirkungen mit umgebenden Wassermolekülen. Trotz seines niedrigen pK a -Werts von 4.6 und einem Abstand von 3.6 zum geladenen Asp189 am Boden der S1-Tasche liegt die Aminogruppe von Anilin protoniert vor.Hingegen wird in 2-Aminopyridin das Pyridin-N-Atom protoniert, obwohl dessen Aminogruppe um 1.6 näher an Asp189 liegt. Folglichi st, abgesehen von Ladungs-Ladungs-Abständen, die Stabilitätd er mçglichen Tautomere ausschlaggebend fürd en erhaltenen Bindungsmodus und damit entscheidend fürdie Vorhersage