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 bean seeds was cloned for structural and biochemical studies. The inhibitor was subjected to two point mutations at a conserved position, Asn14. This residue, known to have a pivotal role in stabilizing the first reactive-site loop (Gln63-Phe68) of the inhibitor, is highly conserved in the sequences of the other members of Kunitz (STI) family as well as in the sequences of Kazal family of serine protease inhibitors. The mutants, N14K and N14D, were subjected to biochemical assay and their characteristics were compared with those of the recombinant inhibitor (rWCI). Crystallographic studies of the recombinant and the mutant proteins are discussed. These studies were primarily aimed at understanding the importance of the protein scaffolding towards the conformational rigidity of the reactive-site loop. Our analysis reveals that, as the Lys14 side chain takes an unusual fold in N14K and the Asp14 side chain in N14D interacts with the loop residues by water-mediated hydrogen bonds, the canonical conformation of the loop has remained effectively intact in both the mutant structures. However, minor alterations such as a 2-fold increase in the inhibitory affinity towards the cognate enzyme were observed.
Ravichandran
S
S
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhan Nagar, Calcutta 700 064, India.
Dasgupta
J
J
Chakrabarti
C
C
Ghosh
S
S
Singh
M
M
Dattagupta
J K
JK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D003001:Cloning, Molecular; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019685:Rosales / Q000737:chemistry; D016415:Sequence Alignment; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C440588:WCI protein, Psophocarpus tetragonolobus; D002918:Chymotrypsin