Bioinformatics is a newly emerging interdisciplinary research area lying at the interface of biological and computational sciences. The computational management of biological information related to genes and its products, whole organism or even ecological systems is crucial not only for gaining an understanding of the molecular basis of biological phenomena but also for the utilization of biological materials. The information so obtained is used in rationalizing protein behaviors, designing experiments that further probe the intimate relationship between structure and function, modify existing proteins or design new proteins, obtain detailed pictures of molecular processes (or diseases), and develop drugs based on chemical similarity of known drugs.
Proteins are essential chemical components of every known form of life. The wide functional ability of these macromolecules arises from nature's ingenuity to build complex structures from simple components. Structural studies on bio-molecules have changed our perception of the biological world in the past twenty years. Today the explanation of a biological function seems incomplete unless the shape of the bio-molecules involved is included in the description. Despite its relevance and importance; bimolecular structure modeling remains one of the most complex and challenging tasks in molecular biology. One technique used to meet this challenge is homology modeling. Homology modeling describes an extended collection of techniques with the goal of predicting 3D details of bio-molecules of unknown structure, relying heavily on resources such as pattern: function relationship and sequence: structure information.
The present study is thus an attempt to gain an understanding of the structure and function of three different protein families namely Granzymes, Caspases and proteinase inhibitors using molecular modeling techniques.
Granzymes are mainly located in the cytotoxic lymphocytes, the key players of cell mediated immunity. These proteins belonging to the family of serine proteases are synthesized as zymogens in the endoplasmic reticulum and converted into active enzymes after a two-step process. The crystal structure of granzyme B from human (Bh) and rat (Br), as well as that of pro-granzyme K (Kh) has been reported recently. In this study, we have attempted to predict the 3D structure of granzyme family (in particular Gzm Ah, Mh, Bm, and Cm from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (Kh), and granzyme B (Bh). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The study also highlights key residues at the s1, s2, and s2' binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, Le., an N-terminal domain and a C-terminal domain comprising predominantly of r3-sheet structure with a little a-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme Bh. For example, in granzyme Mh, valine is present at the s1 sub site instead of arginine. 5imilarly differences at s2 (Leuâ†’Phe), s3 (serâ†’Gly), and S4(Argâ†’Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, serl09), myristoylation (Gly22, 117, and 131), and glycosylation (serl60) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at 5er160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.
Caspases are intracellular cysteine proteinases which playa central role in apoptosis or programmed cell death. Caspases induce apoptosis through a highly integrated and regulated biological, biochemical, and genetic mechanism. Although proper execution of apoptosis is fundamental for cell growth artificial caspase inhibition can be considered in certain degenerative diseases. This realization has attracted attention towards caspases as likely targets for pharmaceutical intervention. We have analyzed the structure of caspase-6, also known as activator caspase, and predict the possible glycosylation, phosphorylation, and myristoylation sites as very little is known about the functional role of these post translational modifications in the caspase family. The predicted tertiary structure of caspase-6 as well as the enzyme complexed with its inhibitor (tetra-peptide aldehyde Ac-IETD-CHO) shows similar binding feature as seen in other caspases. Cys / His catalytic dyad for caspase-6 and -8 show possible involvement of a third component, i.e., Pro29 and Arg258 in caspase-6 and caspase-8, respectively. Changes in the length and nature of loop between as and B9, involved in defining the 54 subsite, result in modification of P4 (lle) site. These interactions provide detail of inhibitor binding on structural level and also help in designing mutants for structure-function studies of these enzymes.
Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymottrypsin. These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against the attack of seed predator. We have also attempted to analyze proteinase inhibitors like (LTI) which shows structural features characteristic of the Kunitz type trypsin inhibitor. LTI consists of 172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI and LTI. Comparison of the active site residues shows that PI-P4 positions of LTI are homologous to STI however the P2' to P4' positions show wide variations. It has been observed earlier that despite structural variations, active site residues in this class of inhibitors are mostly conserved. PI site is Arg which tends to inhibit trypsinlike protease activity. In LTI its scissile site PI-PI' (Arg-lle) is conserved. The PI residues of Arg 62 make the most extensive hydrogen bonds with PPT. The side chain of Arg 62 occupies its expected position in the primary binding pocket of PPT. The guanidinium group of Arg 62 makes an ionic interaction with the carboxylate group of Asp189 in PPT. The phenolic side chain of Tyr61 (P2) is positioned between the side chain of Leu99 and His57, being parallel to the imidazole ring of the latter and its hydroxyl group forms a hydrogen bond with the carbonyl "0" atom of Gly96.
The major contribution in the binding of LTI with the protease molecule comes from eight residues (Thr98, Gln117, Pro60, Tyr61, Arg62, lle63, and Leu64). However, the pattern for the hydrogen bonding interaction involving the reactive site loop residues from Pro (P3) to lIe (Pt' ) is well conserved between the crystal structures STI:PPT and LTI:PPT. We have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique features of LTI.
References: 1. R. Sattar, S. Abid Ali and A. Abbasi. Biochem. Biophys. Res. Commun 308,726-735Ö˝2003 2. Rabia Sattar, S. Abid Ali and Atiya Abbasi Biochem. Biophys. Res. Commun 308 497-504,2003 3. Rabia Sattar, S. Abid Ali, Mustafa Kamal, Aftab Ahmed Khan and Atiya Abbasi Biochem. Biophys. Res. Commun. 314, 755-765, 2003.