Free energy calculations show that acidic P1 variants undergo large pK_a shifts upon binding to trypsin

Abstract

Serine proteinases and their protein inhibitors belong to one of the most comprehensively studied models of protein-protein interactions. It is well established that the narrow trypsin specificity is caused by the presence of a negatively charged aspartate at the specificity pocket. X-ray crystallography as well as association measurements revealed, surprisingly, that BPTI with glutamatic acid as the primary binding (P1) residue was able to bind to trypsin. Previous free energy calculations showed that there was a substantially unfavorable binding free energy associated with accommodation of ionized P1 Glu at the S1-site of trypsin. In this study, the binding of P1 Glu to trypsin has been systematically investigated in terms of the protonation states of P1 Glu and Asp189, the orientation of Gln192, as well as the possible presence of counterions using the linear interaction energy (LIE) approach and the free energy perturbation (FEP) method. Twenty-four conceivable binding arrangements were evaluated and quantitative agreement with experiments is obtained when the P1 Glu binds in its protonated from. The results suggest that P1 Glu is one of the variants of BPTI that inhibit trypsin strongest at low pH, contrary to the specificity profile of trypsin, suggesting a new regulation mechanism of trypsin-like enzymes.

Binding affinities in the SAMPL3 trypsin and host–guest blind tests estimated with the MM/PBSA and LIE methods

“…The protonation state of the ligands were estimated by the Epik software, supplemented by Jaguar calculations for two of the ligands (L15 and L16). Earlier LIE studies have indicated that carboxylate groups bind to trypsin in their protonated (neutral) form [57]. Therefore, we studied the binding of protonated carboxylate groups for ligands L1, L15, and L25, and corrected the binding affinities for the unfavourable protonation of these groups, assuming that the pK a of the free ligand is equal to that of benzoic acid (4.20 [58], i.e.…”

Alchemical free energy simulations for biological complexes: powerful but temperamental …

“…Thus, the comparisons to experiment sometimes test a complex mixture of model predictions, rather than a single, simple prediction. This is analogous to the study of Brandsdal et al, 35 where 24 different states of the trypsin:BPTI system were explored. Similarly, Luzhkov et al…”

“…[18][19][20] Similarly, an approximate, Linear Response approach can be tested by comparison to MDFE, then used to establish protonation or oxidation states of selected groups. [33][34][35] This combination of methods is, arguably, much more powerful than the simple methods alone.To illustrate these points, we use two main data sets. The first, ''medium-throughput'' study considered a dozen variants of the antibiotic tetracycline (Tc), binding to three very different receptors: its target, the 30S ribosomal particle, a resistance protein, the Tet repressor (TetR), and a second, putative target, the protein EF-Tu.…”

“…42 Good agreement with experiment was obtained for the Asp/Asn-binding free energy difference when the predicted His protonation state was used in the MDFE simulations; if another state was assumed, the agreement was very poor 42 ; see next section for more details. A third example is trypsin:BPTI binding, studied recently by Brandsdal et al 35 An Asp-to-Glu mutation was studied with an approximate free energy method, the Linear Interaction Energy (LIE) method, which is analogous to PB/LRA. 23,[27][28][29] The orientation of a nearby Gln and the protonation states of the Asp sidechain were also examined using LIE, whereas the protonation of the mutated Glu was computed with MDFE.…”

Quantitative structure–activity relationship analysis of canonical inhibitors of serine proteases

“…Particular noteworthy are the elegant works that effectively employed linear interaction energy and free energy perturbation methods [6,[11][12][13]. However, to the best of our knowledge, no quantitative structure-activity relationship (QSAR) analysis has been done so far on these very interesting biosystems, in spite of the good and homogeneous experimental data available in the literature.…”