Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics
○Susmita Khamrui Jhimli Dasgupta Jiban K Dattagupta Udayaditya Sen

The scaffold of serine protease inhibitors plays a significant role in the process of religation that resists proteolysis of the inhibitors compared to a substrate. To examine the structural element responsible for 'prevention of proteolysis', we have targeted a conserved scaffolding Asn residue of WCI and mutated it with residues having different shapes and charges like Gly, Ala, Thr, Leu and Gln. Results of structural and biochemical studies performed on these mutants prompted us to conclude that the side chain of spacer Asn not only fits snugly into the concave space of the reactive site loop cavity but also its ND2 atom forms hydrogen bonds with P2 and P1' carbonyl O at either side of the scissile bond holding the cleaved products together for religation. Data base analysis allowed us to identify such spacer asparagines in 5 other families of serine protease inhibitors with similar disposition of their ND2 atom suggesting that Asn mediated religation is prevalent for serine protease inhibitors¹. In another approach, to examine the role of scaffold in broader sense, two chimeric proteins are planned to prepare with loops of trypsin inhibitors like ETI and STI on the scaffold of WCI. As a first step towards this approach, we prepared a P1 mutant (Leu to Arg) of WCI that strongly inhibits trypsin with a Ki value comparable to ETI and STI and structure of this mutant (L65R) at 2.15 A provides a clue to this altered inhibition². More over structure of the complex (2.6 A) between L65R and trypsin provides intricate details of this strong inhibition³. Structural results of the Asn mutants, P1 mutant and complex of it with trypsin along with their biochemical data will be presented here.
Biochemistry (2006) 45:6783
BBA. (2005) 1752:65
Manuscript under preparation