Human leukocyte elastase (HLE) is a serine protease that contributes to tissue destruction in various disease states-for example, in emphysema. FR901277 is a natural product isolated from the culture filtrate of Streptomyces resistomicificus and is a potent inhibitor of both HLE and porcine pancreatic elastase (PPE). FR901277 consists of four normal amino acids and three unusual amino acids, and is a unique bicyclic peptide compound. The crystal structure of PPE complexed with FR901277 has been determined at 1.6 Å resolution. The Oγ atom of Ser-195 in PPE did not form a covalent bond with FR901277, but formed a hydrogen bond with the Nε atom of His-57. On the other hand, the portion from L-Orn(1) through dehydroxyThr(3) in FR901277 formed an antiparallel β-sheet structure with the backbone of the active site in PPE. The S4 through S2' binding subsites in PPE were all occupied by the hydrophobic side chains of the inhibitor molecule. Especially, the ethylidene moiety of FR901277 occupied the S1 specific pocket, indicating a CH/π interaction. In addition, the isopropyl side chain of L-Val(7) was located at the enzyme surface between the S2 and S1' pockets with several van der Waals contacts. However, the amino acid (4) residue was not involved in a significant interaction with PPE. Comparison of inhibitor structures in different environments showed that FR901277 has a highly rigid bicyclic framework; however, it can slightly change its conformation according to the circumstances. The binding mode of FR901277 at the active site of PPE was directly applicable to that in HLE, after consideration of induced fit. The structure of the PPE-FR901277 complex provided much information regarding potential sites for modification of the physicochemical properties of FR901277. (C) 2000 John Wiley and Sons, Inc.; Human leukocyte elastase (HLE) is a serine protease that contributes to tissue destruction in various disease states - for example, in emphysema. FR901277 is a natural product isolated from the culture tiltrate of Streptomyces resistomicificus and is a potent inhibitor of both HLE and porcine pancreatic elastase (PPE). FR901277 consists of four normal amino acids and three unusual amino acids, and is a unique bicyclic peptide compound. The crystal structure of PPE complexed with FR901277 has been determined at 1.6 angstroms resolution. The Oγ atom of Ser-195 in PPE did not form a covalent bond with FR901277, but formed a hydrogen bond with the Nε atom of His-57. On the other hand, the portion from L-Orn(1) through dehydroxyThr(3) in FR901277 formed an antiparallel β-sheet structure with the backbone of the active site in PPE. The S4 through S2′ binding subsites in PPE were all occupied by the hydrophobic side chains of the inhibitor molecule. Especially, the ethylidene moiety of FR901277 occupied the S1 specific pocket, indicating a CH/π interaction. In addition, the isopropyl side chain of L-Val(7) was located at the enzyme surface between the S2 and S1′ pockets with several van der Waals contacts. However, the amino acid (4) residue was not involved in a significant interaction with PPE. Comparison of inhibitor structures in different environments showed that FR901277 has a highly rigid bicyclic framework; however, it can slightly change its conformation according to the circumstances. The binding mode of FR901277 at the active site of PPE was directly applicable to that in HLE, after consideration of induced fit. The structure of the PPE-FR901277 complex provided much information regarding potential sites for modification of the physicochemical properties of FR901277.