Fluorine-induced polarity increases inhibitory activity of BPTI towards chymotrypsin

Leppkes, Jakob; Dimos, Nicole; Loll, Bernhard; Hohmann, Thomas; Dyrks, Michael; Wieseke, Ariane; Keller, Bettina G.; Koksch, Beate

doi:10.1039/d2cb00018k

Cited by 11 publications

(17 citation statements)

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“…Having synthetic access to the monofluorinated amino acid MfeGly and the corresponding variant of BPTI, we now determined the crystal structure of the Lys15MfeGly–BPTI−β-trypsin complex and deposited it in the Protein Data Bank (pdb: 7PH1). The crystal structure shows five water molecules in the binding pocket (Figure ).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2022

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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 We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly).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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2022

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“…The replacement of a single C–H bond with C–F is generally considered to be isosteric. 20,21 Investigations of the effects of fluorinated amino acids on hydrophobicity, 22,23 secondary structure formation, 24,25 protein–protein interactions, 26,27 amyloid folding kinetics, 28,29 proteolytic stability, 30 the chemical and biological properties of fluorinated peptide-based materials, 31 and the integration of fluorine into bacteria 32,33 have been reported by our group. The vast majority of previous studies including our own efforts examining fluorinated amino acids in the context of peptide and protein chemistry were limited to the incorporation of one or only a few of these building blocks.…”

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confidence: 97%

Rational design of amphiphilic fluorinated peptides: evaluation of self-assembly properties and hydrogel formation

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“…Having synthetic access to the monofluorinated amino acid MfeGly 37 and the corresponding variant of BPTI 38 , we now determined the crystal structure of the Lys15MfeGly-BPTI- β -trypsin complex and deposited it in the Protein Data Bank (pdb: 7PH1). The crystal structure shows five water molecules in the binding pocket (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2.a shows the structure of the wild-type(wt)-BPTI in complex with β-trypsin 18 . βtrypsin has a deep S1 binding pocket, and the catalytic triad is located at the rim of this Having synthetic access to the monofluorinated amino acid MfeGly 37 and the corresponding variant of BPTI 38 , we now determined the crystal structure of the Lys15MfeGly-BPTIβ-trypsin complex and deposited it in the Protein Data Bank (pdb: 7PH1). The crystal structure shows five water molecules in the binding pocket (Fig.…”

Section: B Inhibition Assay and Binding Constantsmentioning

confidence: 99%

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

Wehrhan

Leppkes

Dimos

et al. 2022

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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 act can form specific non-covalent interactions to 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 Lys15MfeGly-BPTI-β-trypsin complex (pdb: 7PH1). Both, experimental and computational data, show a step-wise 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 regards to energetic properties that could explain the increase in inhibitor strength. The analysis of the enthalpy 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.

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Fluorine-induced polarity increases inhibitory activity of BPTI towards chymotrypsin

Abstract: Substituting the P1 position in bovine pancreatic trypsin inhibitor (BPTI) is known to heavily influence its inhibitory activity towards serine proteases. Side-chain fluorinated aliphatic amino acids have been shown to...

Cited by 11 publications

References 58 publications

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

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

Rational design of amphiphilic fluorinated peptides: evaluation of self-assembly properties and hydrogel formation

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

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