Water Network in the Binding Pocket of Fluorinated BPTI–Trypsin Complexes─Insights from Simulation and Experiment

Abstract

Structural waters in the S1 binding pocket of β-trypsin are critical for the stabilization of the complex of β-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly–BPTI−β-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly.

Pre-bound State Discovered in the Unbinding Pathway of Fluorinated Variants of the Trypsin-BPTI Complex Using Random Acceleration Molecular Dynamics Simulations

“…We ran all Molecular Dynamics (MD) simulations using Gromacs [28][29][30] software and our self-parameterized Amber14SB force field 21,[31][32][33] . Energy minimizations were conducted with the steepest descend algorithm.…”

Catalytically Competent Fluorinated Barnase Variants

“…This particular family of building blocks has also been shown to restore inhibitory activity to bovine pancreatic trypsin inhibitor substituted at a key lysine position with Abu. 7 8 9…”

“…Our group has found significant effects of mono-to trifluorinated side chains based on aminobutyric acid (Abu) on, for example, the rate of amyloid formation 5 , and proteolysis 6 in peptides. This particular family of building blocks has also been shown to restore inhibitory activity to bovine pancreatic trypsin inhibitor substituted at a key lysine position with Abu [7][8][9] .…”

“…This particular family of building blocks has also been shown to restore inhibitory activity to bovine pancreatic trypsin inhibitor substituted at a key lysine position with Abu. [7][8][9] Here we present a straightforward microwave-assisted solid-phase peptide synthesis (MW-SPPS) protocol for the full-length synthesis of the wild-type ribonuclease barnase and three fluorinated analogues substituted at position 27 of the sequence. Barnase and complexes between it and its inhibitor barstar have been very well characterized enzymatically and spectroscopically.…”

Free Energy Density of a Fluid and Its Role in Solvation and Binding

“…Approaches to the spatial mapping of solvation thermodynamics include inhomogeneous solvation theory (IST), − which underpins the WaterMap, − STOW, Grid Inhomogeneous Solvation Theory (GIST), − and related , technologies, classical density functional theory (DFT), − Grid-Cell Theory (GCT), the three-dimensional reference interaction site model (3D-RISM), − and others, − as recently reviewed . Such spatial decompositions have been of particular interest in studies of the physical chemistry of noncovalent binding ,− and as guides to host–guest chemistry and structure-based drug design. − For example, a ligand that displaces thermodynamically unfavorable water from a receptor site is expected to bind the receptor more tightly than one that does not, other things being equal. ,, Alternatively, a modification to the ligand that stabilizes water within the site may enhance binding. ,− This body of work assumes that one can meaningfully discuss the local densities of free energy, enthalpy, and entropy in a fluid and that concepts such as “unfavorable”, “disfavored”, “unstable”, “high energy”, or “low entropy” water are well-founded in theory.…”