Understanding Binding Selectivity toward Trypsin and Factor Xa: the Role of Aromatic Interactions

Abstract

A congeneric series of four bis-benzamidine inhibitors sharing a dianhydrosugar isosorbide scaffold in common has been studied by crystal structure analysis and enzyme kinetics with respect to their binding to trypsin and factor Xa. Within the series, aromatic interactions are an important determinant for selectivity discrimination among both serine proteases. To study the selectivity-determining features in detail, we used trypsin mutants in which the original binding site is gradually substituted to finally resemble the factor Xa binding pocket. The influence of these mutations has been analyzed on the binding of the closely related inhibitors. We present the crystal structures of the inhibitor complexes obtained by co-crystallizing an "intermediate" trypsin mutant. They could be determined to a resolution of up to 1.2 A, and we measured the inhibitory activity (K(i)) of each ligand against factor Xa, trypsin, and the various mutants. From these data we were able to derive a detailed structure-activity relationship which demonstrates the importance of aromatic interactions in protein-ligand recognition and their role in modulating enzyme selectivity. Pronounced preference is experienced to accommodate the benzamidine anchor with meta topology in the S(1) specificity pocket. One ligand possessing only para topology deviates strongly from the other members of the series and adopts a distinct binding mode addressing the S(1)' site instead of the distal S(3)/S(4) binding pocket.

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

“…These different hydrophobically modified residues bind into the hydrophobic S3/4 pocket of trypsin and are responsible for the observed increasing affinity. This pocket is supposed to favor binding of ligands possessing bulky aromatic moieties [51].…”

Human trypsinogens in the pancreas and in cancer

“…The aromatic character of this pocket increases from trypsin (only Trp215) to factor Xa (Trp215, Tyr99, Phe174). The results show that the establishment of favourable directional aromatic-aromatic interactions in the S3/S4 pocket with a bound ligand will increasingly contribute to binding affinity and will thus determine selectivity (Di Fenza et al, 2007). Factor Xa is thus more selective with respect to bovine trypsin for ligands which opportunely interact with the fully established aromatic box in the S3/S4 subsite.…”

“…Contribution of aromatic interactions in binding affinity and ligand selectivity in the S3/ S4 pocket of bovine trypsin, human factor Xa and chimeric S3/S4 mutants have been studied (Di Fenza et al, 2007). The aromatic character of this pocket increases from trypsin (only Trp215) to factor Xa (Trp215, Tyr99, Phe174).…”

Prediction of trypsin/molecular fragment binding affinities by free energy decomposition and empirical scores

“…To assess the accuracy of pose generation and the general protocol for calculating binding affinities, we constructed a small training set, which consisted of known binders of trypsin. [43], and 1Y5B [44]. These ligands covered a range of molecular weights from 122 to 525 g/mol and they spanned a range of binding affinities from 36 mM to 40 nM.…”