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.