Structure-function relationship of serine protease-protein inhibitor interaction

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases . Recently, we have determined high resolution solution structures of two i nhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitat issimum trypsin inhibitor (LUTI) in the free state and an ultra high resolu tion X-ray structure of BPTI. All three inhibitors, despite totally differe nt scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calorimetry d ata show that the interaction between wild type BPTI and chymotrypsin is en tropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from th e protease binding loop of BPTI: P-1, P-1', P-3, and P-4. We mutated these residues to different amino acids and the variants were characterized by de termination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P-1 residu e in the S-1 pocket of four proteases: chymotrypsin, trypsin, neutrophil el astase and cathepsin G was probed with 18 P-1 variants. High resolution X-r ay structures of ten complexes between bovine trypsin and P-1 variants of B PTI have been determined and compared with the cognate P-1 Lys side chain. Mutations of the wild type Ala16 (P-1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P -1' position leads to steric conflicts in the vicinity of the mutation. Fin ally, mutations at the P-4 site allowed an improvement of the association w ith several serine proteases involved in blood clotting. Conversely, introd uction of Ser, Val, and Phe in place of Gly12 (P-4) had invariably a destab ilizing effect on the complex with these proteases.