Redesigning the reactive site loop of the wheat subtilisin/chymotrypsin inhibitor (WSCI) by site-directed mutagenesis. A protein-protein interaction study by affinity chromatography and molecular modeling.
A site-directed mutagenesis strategy was employed to obtain four mutants of wheat subtilisin/chymotrypsin inhibitor (WSCI), with the aim to produce inactive forms of this protein. The mutants were expressed in Escherichia coli as fusion proteins and, after the tag removal, were purified to homogeneity. Three mutants, containing a single mutation at the sequence positions 49 or 50, were named E49S, E49P and Y50G, respectively. These mutants exhibited anti-subtilisin activities comparable to that of the wild type protein; instead, anti-chymotrypsin activity was detectable only for the mutant E49S. A fourth mutant (M48P-E49G), containing a double amino acid substitution at the inhibitor reactive site (P1-P1'), was inactive against both subtilisin and chymotrypsin. In order to investigate the interactions between the putative susceptible enzymes and the mutated forms of WSCI, we performed time-course hydrolysis experiments by incubating samples of the mutants with subtilisin-agarose and chymotrypsin-agarose, respectively. These experiments yielded information on the E/I complex formation, as well as on the timing of the cleavage pattern of some of these mutants. Molecular modeling studies were carried out with the 3D models of the mutants and of their putative complexes with subtilisin and chymotrypsin. In terms of inter- and intra-chain H-bond networks, the observations made for each theoretical E/I complex were found to be fully coherent with experimental data (kinetic and time-course hydrolysis) and supplied specific modalities of interaction of each mutant with the enzyme counterpart.
Bruni
Natalia
N
Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, I-01100 Viterbo, Italy.
Di Maro
Antimo
A
Costantini
Susan
S
Chambery
Angela
A
Facchiano
Angelo M
AM
Ficca
Anna Grazia
AG
Parente
Augusto
A
Poerio
Elia
E
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014908:Triticum / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D011994:Recombinant Proteins; C504099:WSCI protein, Triticum