Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of alzheimer's amyloid β‐protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): Engineering of inhibitors with altered specificities

Abstract

The crystal structures of the inhibitor domain of Alzheimer's amyloid @-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (PI), residue 15(I)' is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched PI side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15. 17, and 19, which are major binding determinants. Inhibitor variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.

Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…8)-In addition to PN2KPI loop 1 amino acids (Figs. 5-7), the loop 2 region amino acid, Phe 34 , was also replaced with Ala, resulting in an 8-fold loss of activity (IC 50 of 9.85 nM) as compared with WT PN2KPI (IC 50 of 1.28 nM), which agrees with the loss of activity in the APTT assay (Fig. 8, A and B).…”

“…After surveying all of the serine protease structures in complex with APPI and BPTI in the Protein Data Bank, we observed that Glu/Gln 192 of the serine protease forms either no hydrogen bond or one hydrogen bond with the carbonyl oxygen atom of Cys 14 (P2) of APPI or BPTI. In the structure of FVIIa with a BPTI mutant (Protein Data Bank code 1FAK (37)), which mimics the first Kunitz domain of tissue factor pathway inhibitor (the natural inhibitor of the tissue factor FVIIa complex), Lys 192 is hydrogen-bonded to Asp 11 (a mutated residue, T11D) and the carbonyl oxygen atom of Tyr 34 (V34Y) of the BPTI mutant. We suspect that Lys 192 plays an important role in FXIa activity and is essential for the interaction between FXIa and PN2KPI.…”

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

“…4, intact APPI is a 57-amino acid peptide featuring three internal disulfide bonds and the N-terminal sequence EVCSE. The reactive site bond identified in crystal structures of APPI bound to FXIa (33) and trypsin (39,40) is located between Arg 13 and Ala 14 (indicated by a black arrow in Fig. 4); from here on, we will refer to this bond as the Arg 15 -Ala 16 bond, according to its alignment with the archetypal Kunitz family inhibitor BPTI and for consistency with the bulk of prior literature.…”

“…tive site of APPI. Crystal structures indicate that the majority of contacts between APPI and trypsins are formed by the P 4 -P 3 Ј residues Gly-Pro-Cys-Arg-Ala-Met-Ile (39,40). We synthesized a peptide recapitulating this sequence, but substituting the P 2 Cys with Ser to prevent peptide dimerization and adding a Tyr residue at the P 4 Ј C terminus to facilitate peptide quantification.…”

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

“…The bovine pancreatic trypsin inhibitor belongs to the Kunitz-type inhibitors and is a potent inhibitor of serine proteases. The crystal structures of BPTI with bovine trypsin and with bovine chymotrypsin have been determined [47][48][49]. Analysis of these complexes have shown that there are 10-13 residues on the inhibitor forming less than 4 Å contacts with 20-25 residues of the enzyme.…”