Engineered Eglin c Variants Inhibit Yeast and Human Proprotein Processing Proteases, Kex2 and Furin

Abstract

We engineered eglin c, a potent subtilisin inhibitor, to create inhibitors for enzymes of the Kex2/furin family of proprotein processing proteases. A structural gene was synthesized that encoded "R(1)-eglin", having Arg at P(1) in the reactive site loop in place of Leu(45). Ten additional variants were created by cassette mutagenesis of R(1)-eglin. These polypeptides were expressed in Escherichia coli, purified to homogeneity, and their interactions with secreted, soluble Kex2 and furin were examined. R(1)-eglin itself was a modest inhibitor of Kex2, with a K(a) of approximately 10(7) M(-)(1). Substituting Arg (in R(4)R(1)-eglin) or Met (in M(4)R(1)-eglin) for Pro(42) at P(4) created potent Kex2 inhibitors exhibiting K(a) values of approximately 10(9) M(-)(1). R(4)R(1)-eglin inhibited furin with a K(a) of 4.0 x 10(8) M(-)(1). Introduction of Lys at P(1), in place of Arg in R(4)R(1)-eglin reduced affinity only approximately 3-fold for Kex2 but 15-fold for furin. The stabilities of enzyme-inhibitor complexes were characterized by association and dissociation rate constants and visualized by polyacrylamide gel electrophoresis. R(4)R(1)-eglin formed stable 1:1 complexes with both Kex2 and furin. However, substitution of Lys at P(2) in place of Thr(44) resulted in eglin variants that inhibited both Kex2 and furin but which were eventually cleaved (temporary inhibition). Surprisingly, R(6)R(4)R(1)-eglin, in which Arg was substituted for Gly(40) in R(4)R(1)-eglin, exhibited stable, high-affinity complex formation with Kex2 (K(a) of 3.5 x 10(9) M(-)(1)) but temporary inhibition of furin. This suggests that enzyme-specific interactions can alter the conformation of the reactive site loop, converting a permanent inhibitor into a substrate. Eglin variants offer possible avenues for affinity purification, crystallization, and regulation of proprotein processing proteases.

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation

“…harose chromatography essentially as previously described (18). Eglin c was dialyzed into 10 mM (NH 4 ) 2 OAc at pH 6.0, lyophilized, and stored at Ϫ80°C until use, when it was reconstituted with H 2 O. Crystallization-A 1:1 (mol/mol) mixture of CTRC and eglin c was concentrated to achieve a protein concentration of 4 -5 mg/ml.…”

“…In approximately half of the families affected by autosomal dominant hereditary pancreatitis, the disease is caused by mutations in the cationic trypsinogen gene PRSS1 that result in either enhanced trypsinogen activation or resistance to degradation (2)(3)(4). CTRC possesses the unique capacity to impact trypsinogen activation and stability via two opposing mechanisms: it can cleave cationic trypsinogen either at Phe 18 -Asp 19 within the trypsinogen activation peptide, leading to enhanced autoactivation (5), or at Leu 81 -Glu 82 within the Ca 2ϩ -binding loop, resulting in degradation (6). A number of disease-causing cationic trypsinogen mutations exert their effect in part through accelerating cleavage by CTRC at Phe 18 -Asp 19 or through diminishing cleavage by CTRC at Leu 81 -Glu 82 (4).…”

“…CTRC possesses the unique capacity to impact trypsinogen activation and stability via two opposing mechanisms: it can cleave cationic trypsinogen either at Phe 18 -Asp 19 within the trypsinogen activation peptide, leading to enhanced autoactivation (5), or at Leu 81 -Glu 82 within the Ca 2ϩ -binding loop, resulting in degradation (6). A number of disease-causing cationic trypsinogen mutations exert their effect in part through accelerating cleavage by CTRC at Phe 18 -Asp 19 or through diminishing cleavage by CTRC at Leu 81 -Glu 82 (4). The p.A16V mutation, which accounts for a small percentage of hereditary pancreatitis kindreds and is also associated with idiopathic chronic pancreatitis (7,8), appears to exert its pathological effect solely by increasing the vulnerability of the cationic trypsinogen activation peptide to cleavage by CTRC (4).…”

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

“…An examination of physiological Kex2 substrates does not indicate any obvious P 6 selectivity, and in experiments with peptide substrates, Kex2 exhibits only a 2-fold preference for Arg at P 6 (14). This difference in P 6 recognition was also observed in interactions with derivatives of eglin-c that had been engineered to be potent inhibitors of Kex2 and furin (19). Kex2 exhibited only a slightly higher (ϳ3-fold) affinity for Arg (as opposed to Gly) at P 6 in an eglin-c variant having Arg at P 1 and P 4 (19).…”

Development of Protein-based Inhibitors of the Proprotein of Convertase SKI-1/S1P

“…The various successful approaches include: active site-directed chloromethyl ketone inhibitors (12,13), reversible peptide-based inhibitors (14 -17), plant derivatives (18), and several engineered variants of protein-based inhibitors that possess a furin-like motif. These include ␣2-macroglobulin (␣2-MF) (19), ␣ 1 -antitrypsin (␣ 1 -AT) Portland (␣ 1 -PDX) (20 -22), proteinase inhibitor 8 (PI8) (23), the turkey ovomucoid third domain (24), and eglin C (25,26). However, these effective inhibitors directed against the basic amino acid-specific members lack selectivity.…”