Molecular Simulation Study On The Interactions Between Chymotrypsin And Guamerin

As part of protein–protein recognition, the precise recognition between protease and its protein substrate is also responsible for the control of multiple physiological processes, such as virus maturation, pro-hormone processing, blood coagulation, apoptosis and signal transduction.The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. bovine chymotrypsin is particularly suitable for specificity analysis because its kinetic properties towards low molecular mass substrates has been described using a vast amount of data in Previous work. The S1 binding pocket of bovine chymotrypsin, responsible for the accommodation of the substrate P1 residue, constitutes the main specificity determinant of this enzyme.Elastases catalyze the cleavage of fibrous elastin, an important structural protein required for the elasticity of tissues and organs.1 Elastin is predominant in the lungs, arteries, skin and ligaments, and the imbalance between elastase and its natural inhibitors is associated with tissue damage and can cause various diseases in lung and connective tissues, such as respiratory distress syndrome, pulmonary emphysema, asthma, cystic fibrosis, rheumatoid arthritis and infectious diseases. Furthermore, excess neutrophil elastase might be involved in cancer risks as well as in the development of acute promyelocytic leukemia.4 Therefore, it is thought that the development of elastase-specific inhibitors will enable us to control elastase-associated diseases. In this regard, there have been efforts to develop elastase inhibitors using endogenous peptides such as secretory leukocyte protease inhibitor5 and elafin.6 Peptide inhibitors identified from diverse natural sources including soybean, turkey ovomucoid and leech have also been tested for the same purpose.Guamerin, a 57-amino-acid polypeptide having a molecular weight of 6110 D, was isolated from the leech Hirudo nipponia. It shows inhibition constant (Ki) of 8.1×10-14M.It is stable at a wild range of pH from 1 to11 and heat-stable up to 90℃. It is a cysteine-rich serine proteinase inhibitor with a identical spacing of 10 cysteine residue, which shows no similarity to any known elastase inhibitors but has 48% sequence homology to hirustasin belongs to the antistasin-type serine proteinase inhibitor family. Indeed, Guamerin shares many distinctive structural features with the members of the antistasin family inhibitors including the overall structural topology, the order of disulfide cross-links, the active-site binding loop, and the orientation of P1 residues (Fig. 1a and b). Guamerin also overlaps well with other inhibitors belonging to the same family such as hirustasin and bdellastasin with RMSDs of 1.9 and 1.0 ? for 46 Cαatoms, respectively.Guamerin was originally characterized as an elastase-specific inhibitor for its ability to inhibit human leukocyte and porcine pancreatic elastases (PPEs). Because the relative inhibitory activity of Guamerin against other proteases was reported to be less than 10% of the activity against elastase,though it also shows inhibitory activity against other proteases. High inhibitory activity of Guamerin against human leukocyte and porcine pancreatic elastases (PPEs) make it possible to apply it to various diseases caused by elevated elastase concentration.However, in Kim's study, the recombinant Guamerin produced in Escherichia coli is not specific to elastase, but rather is specific to chymotrypsin. Its inhibitory activity against BPC(bovine pancreatic chymotrypsin) is three fold higher than that against PPE. That means, at least, Guamerin inhibits both elastase and chymotrypsin strongly and this limit the application of Guamerin.As a serine protease inhibitor, Guamerin binds to the hydrophobic pocket of the protease in a substrate-like manner using its binding loop. Like other members of serine protease inhibitor family, different residues at the P1 position of Guamerin may also result in magnitude difference of the association constant values with the protease. The interaction between the bovine chymotrypsin and Guamerin, which belongs to the bovine chymotrypsin–serine protease inhibitor interaction, could also be a new good studied model of the interaction and it would benefit the study of both chymotrypsin and Guamerin.For PPE shows high sequence and structural homologies with BPC, and the active sites of PPE and BPC superimpose well, too. In order to improve the binding selectivity of Guamerin to elastase, with these help of the crystal structure of Guamerin in complex with bovine chymotrypsin (BPC) determined by themselves, Kim et al inferred the binding mode of Guamerin to PPE, mutated several residues in the binding loop of Guamerin to a set of natural amino acid residues and successfully achieve their purpose at last. The substitution of the Met36 residue for Ala in the P1 position increased the inhibitory activity against elastase up to 14-fold, the same mutant showed 7-fold decreased activity against chymotrypsin compared to the wild-type Guamerin at the same time. In addition, many valuable information has been obtained from the crystal structure of Guamerin in complex with bovine chymotrypsin (BPC), for instance, Guamerin is composed ofβstrands and loops that are stabilized by disulfide bonds.The experiment results show that the alteration of P1 Met to Ala in Guamerin would cause loss of inhibitory activity against chymotrypsin. And this conclusion was also in accordance with a previous report.15That is, methionine is commonly found as the P1 residue in many inhibitors and exerts strong inhibitory activity against chymotrypsin.In order to get the changes of phenomena in micro-level, which is hard to get in traditional experiment, we could explore the bovine chymotrypsin–Guamerin interaction by molecular dynamics simulation. For it has been a useful tool in many areas, it would help us understand the inhibitory mechanism of Guamerin against chymotrypsin in a molecular level.Here, we employ the crystal structure of Guamerin in complex with bovine chymotrypsin (BPC) as the starting structure of the wild-type after modeling the missing residue,and alter the P1 site residue Met to Ala with software SPDBV in order to get the M36A mutation. In our work a simulation with the time of 30 ns had been carried out, respectively, for the both wild type and M36A mutation. And the simulations show that the alteration of P1 Met to Ala decrease flexibility of Guamerin obviously, particularly, flexibility decrease sharply in N-term Arm. In contrast, the binding loop changes slightly whether in stability or in flexibility. However, the mutation of P1 site changes the binding mode of complex due to the lightly similar internal motions. The changes of interaction energy and flexibility are in accordance with the inhibitory activity. In a word, the possible reason of the loss of activity would be the changes in flexibility of N-Terminal fragment of Guamerin.