Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

Abstract

The Tyr35→Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by x-ray crystallography and isothermal titration calorimetry. The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 Å resolution x-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 Å data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical to that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Abbreviations used: BPTI, bovine pancreatic trypsin inhibitor; Amino acid replacements are indicated by the wild-type residue type (using the one letter code for the 20 standard amino acid residues), followed by the residue number and the mutant residue type; Elsewhere, amino acid residue types are indicated by the standard 3-letter code. IAS, iso-aspartate; HEPES, N -2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)-aminomethane; NMR, nuclear magnetic resonance.

Mechanisms and specificity of factor XIa and trypsin inhibition by protease nexin 2 and basic pancreatic trypsin inhibitor

“…2A. For comparison of three-dimensional structures, we superimposed the PN2KPI X-ray crystal structure from the PN2KPI-FXIa catalytic domain complex (PDB:1ZJD) (28) and the BPTI structure from the BPTItrypsin complex (PDB: 2FTL) (29). The crystal structures of these two proteins were superimposable, displaying a striking similarity of the main-chain conformations of PN2KPI and BPTI.…”

“…The rationale for the present study, which focused on a comparison of the mechanisms of inhibition of human FXIa and bovine trypsin by the human PN2KPI and BPTI, is based on the striking structural homology between the human FXIa/PN2KPI complex (28) and the bovine trypsin/ BPTI complex (29), in addition to the fact that BPTI (as Trasylol R ) is frequently utilized therapeutically in human subjects in the treatment and prevention of thrombo-embolism, e.g. in patients undergoing cardiopulmonary bypass surgery (30,31).…”

“…However, the shorter Lys side chain (compared to Arg) relies on an ordered water molecule to establish the Lys 15 -Asp 189 interaction in the BPTItrypsin complex (Fig. 2D), (PDB:2FTL) (29). Thus, whereas PN2KPI and BPTI have similar backbone structures and P1 residue side chain orientations, differences in side chain bulkiness, length and consequent charge distribution, result in significant differences in local intermolecular interactions with FXIa.…”

Sequence and Conformational Specificity in Substrate Recognition

“…In the context of the Kunitz domain fold, the canonical binding loop is rigidly structured, with mobility limited by the compact packing of the protein, by a disulfide bond that links the P 2 residue to the scaffold, and by hydrogen bonds that tether the backbone of PЈ 1 and PЈ 3 residues to the scaffold. This conformational stability of the inhibitory loop is believed to contribute to the unusually high affinity interactions of canonical inhibitors toward cognate serine proteases (18), and mutations that confer increased flexibility on the canonical loop also diminish affinity toward target proteases (44). By contrast, the canonical-like loop of E-selectin is minimally restrained by intramolecular contacts, and very high B-factors in this region of E-selectin crystal structures suggest that the loop is highly flexible (42).…”

“…6B). Although our two structures represent enzyme complexes with good substrates by kinetic criteria, and not mesotrypsin inhibitors, the orientations of reactive site bonds are indistinguishable from those observed in canonical inhibitor complexes with other trypsins, such as the complex of bovine trypsin with BPTI (PDB entry 2FTL) (44) (Fig. 7A).…”