The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14{->}Lys and Asn14{->}Asp

A double-headed chymotrypsin inhibitor, WCI, from winged beanseeds was cloned for structural and biochemical studies. Theinhibitor was subjected to two point mutations at a conservedposition, Asn14. This residue, known to have a pivotal rolein stabilizing the first reactive-site loop (Gln63–Phe68)of the inhibitor, is highly conserved in the sequences of theother members of Kunitz (STI) family as well as in the sequencesof Kazal family of serine protease inhibitors. The mutants,N14K and N14D, were subjected to biochemical assay and theircharacteristics were compared with those of the recombinantinhibitor (rWCI). Crystallographic studies of the recombinantand the mutant proteins are discussed. These studies were primarilyaimed at understanding the importance of the protein scaffoldingtowards the conformational rigidity of the reactive-site loop.Our analysis reveals that, as the Lys14 side chain takes anunusual fold in N14K and the Asp14 side chain in N14D interactswith the loop residues by water-mediated hydrogen bonds, thecanonical conformation of the loop has remained effectivelyintact in both the mutant structures. However, minor alterationssuch as a 2-fold increase in the inhibitory affinity towardsthe cognate enzyme were observed.