Proteolytic enzymes control many physiological responses. They play a key role in the turnover of proteins and provide a level of quality control for the cellular protein pool by eliminating abnormal and potentially toxic proteins. They are capable of processing misfolded and denatured proteins. The identification and characterization including structure-function relationship studies of specific proteases offer the prospect of new medical advances.
The present study describes the homology modeling studies of different proteinases and polypeptide serine proteinase inhibitors have been performed to trace structure-function relationships. These homology models have proved helpful in proposing hypotheses about the location of substrate/inhibitor-binding sites, substrate specificity and structure-based ligand design. Homology modeling of these proteins also explained some experimental observations from the structural standpoint. During these investigations protein sequences of different length and different similarities to st11lcturally characterized proteins were used to construct the homology models.
Sequences of human Procathepsin E, a novel cysteine proteinase CED3 protein from nematode Caeenrhabditis elegans, three snake venom kunitz type polypeptide inhibitors and two cathepsin B from chicken and hookworm were used as targets for three dimensional structure prediction by homology modeling.
A summary of these studies is given bellow.
Cathepsin E is an intracellular, non-lysosomal, non-secretory aspartic proteinase. In human red cell the aspartic protease cathepsin E is found largely in the membrane-associated zymogen from e.g. Procathepsin E. A three dimensional structural model of human Procathepsin E has been constructed based upon the crystal structures of porcine pepsinogen. The overall protein folding features of the model are similar to those observed in the template structures. The propeptide packs into the active site cleft with a similar secondary structural pattern and associated with enzyme segment by salt-bridges, hydrogen bon dings and hydrophobic interactions. As judged from the model, the salt bridges present between the propeptide and enzyme segment show remarkable variations compared to porcine pepsinogen and human progastricin structures. Mapping of these interactions revealed that human Procathepsin E might engage a different structural motif (a-helix; 12P-19P) for protecting/blocking of catalytic site compared to pepsinogen and progastricin.
CE03 protein, the product of a gene necessary for programmed cell death in the nematode Caenorhabditis elegans, is related to a highly specific cysteine protease family e.g. caspases. A three-dimensional homology model of the complex of CE03-protein with a potent tetrapeptide-aldehyde inhibitor has been calculated based upon the X-ray structure of apopain (caspase-3). As judged from the model, the conformation 0: CE03 protein substrate-binding site and the general binding features of inhibitor at substrate-binding site are similar to that observed in apopain. The tight binding of P I position aspartic acid at the S I subsite is conserved with somewhat different set of interactions compared to known interleukin-lβ converting enzyme (ICE) and apopain structures. The P4 side chain & accommodated well in the S4 site of CE04 protein, however it binds in a different manner for the two enzymes. The modeling indicated that although the CE03 protein needs aspartic acid at both P I and P4 positions for substrate recognition (as evident from studies defining substrate specificities), still variations occur in the binding of these residues at their respective subsites in the enzymes. This model allowed the definition of substrate specificities of CE03 protein on structural stand point which may help in the design of mutants for structure-function studies of this classical caspase homologue.
SNAKE VENOM SERINE PROTEASE INHIBITORS AND THEIR INTERACTIONS WITH TRYPSIN AND CHYMOTRYPSIN
Three homology models of trypsin and chymotrypsin inhibitor polypeptides from snake venom of Naja naja naja and Leaf-nosed viper in the unbound state and in complex with trypsin and chymotrypsin were built based on homology to bovine pancreatic trypsin inhibitor (BPTl). These venom inhibitors belong to the Kunitz-type inhibitor family, which is characterized by a distinct tertiary fold with three-conserved disulfide bonds. The general folding pattern in these trypsin and chymotrypsin inhibitor homology models is conserved when compared to BPTI. The respective orientations of the inhibitors bound to trypsin/chymotrypsin are similar to that of BPTI bound to bovine trypsin/chymotrypsin. The principal binding loop structure of the inhibitors fills the active site of enzymes in a substrate-like conformation and forms a series of independent main-chain and side-chain interactions with enzymes. In order to provide the possible fingerprints for molecular recognition at the enzyme-inhibitor interface, a detailed theoretical analysis of the interactions between the principal binding loop of these inhibitors and active site of trypsin/chymotrypsin is performed based on available crystal structural, site-directed mutagenetic, kinetic and sequence analysis studies.
Despite the variations present at different positions of the principal binding loop of trypsin and chymotrypsin inhibitor models from Leal-nosed viper and cobra Naja naja naja respectively (designated as LnvTI and NCI), there are favorable subsite binding interactions which are expected to exhibit equally potent inhibitory activity as BPTI. On the contrary, significant mutations at several secondary specificity positions 111 the Naja naja naja tIypsin inhibitor (designated as NTI) are likely to affect different inhibitor-enzyme-subsites interactions. This may explain the observed increased inhibitory activity of this polypeptide on structural basis.
CATHEPSIN B FROM CHICKEN AND HOOKWORM
Knowledge based comparative models of chicken cathepsin B and cathepsin B like enzyme acc1 from hookworm Ancylostoma caninun were constructed by using X-ray crystal structural data of human cathepsin B to identify structural features that determine substrate binding and specificity. The general folding of the predicted models is conserved with respect to the human template. The active site of chicken cathepsin B model has similar subsite binding geometry and is expected to exhibit typical cathepsin B-like catalytic activity. In contrast, mutations at different subsites of the active center. of enzyme from hookworm, will most likely result in differences in substrate binding specificity as regards to its human parallel. The structural features of the active site model from hookworm revealed possible cathepsin L-like activity despite its cathepsin B-like primary and suggested tertiary structure.