Book of Abstracts: Albany 2003
June 17-21 2003
Insight to Structural Subsite Recognition in Plant Thiol Protease?inhibitor Complexes: Understanding the Basis of Differential Inhibition and the Role of Water
This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors leupeptin, E-64, E-64-C, ZPACK and homologous plant cysteine proteases (actinidin, caricain, chymopapain, calotropin D) of papain family with a view to understand the various interactions, nature of water binding, factors influencing it and the structural basis of differential inhibition. The tertiary structure of the enzyme-inhibitor complexes were built by interactive modeling and were subjected to energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the contribution of potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are quite similar, critical inspection of the model complexes reveal some significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to alteration in residues arising from sequence differences in the equivalent positions of respective subsites leading to differential inhibition. The key finding of the present study is a conserved site of a water molecule near oxyanion hole of the enzyme active site which is found in all the modeled complexes as well as in several solved crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding site of these homologous proteins suggest the structural importance of the water, which could effectively change the conventional definition on chemical geometry of inhibitor binding domain by changing its shape thus affecting the complimentarity. Again, the water mediated recognition of inhibitor to enzyme subsites (Pn?H2O?.Sn) of leupeptin acetyl oxygen in caricain and chymopapain may also be an additional information, which could offer valuable insight to potent inhibitor design. The spectroscopic characterization (for big inhibitor molecule e.g. cystatin) has also been done using fluorescence, circular dichroism, UV spectrophotometric methods. These studies reveal that the environments of the aromatic amino acid residues of the active site of the protease and the inhibitors have been changed during complexation.
Stereoscopic view of probable binding mode of E-64-C with the active site residues of Actinidin