Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations

Abstract

Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity

“…The degree of flexibility of the region containing the inhibitor reactive site is essential for the productive interaction between this peptide bond and the catalytic site of the target/cognate enzymes. 12 In the present study we postulate that WSCI could represent a versatile scaffold to develop recombinant proteins exhibiting enhanced anti-proteinase activities or new-acquired specificities. The genes coding for these novel inhibitors could be employed for the transformation of non-food use plants, in order to improve their resistance to the attacks of phytophagous insects and microorganisms.…”

“…Similarly to what previously described for the interaction of muteins M48K and M48K-E49D with trypsin, the mutein E49D eluted as an intact molecule from the column during the acidic elution; such behaviour was evidenced even after 60 min of incubation (RP-HPLC analysis, data not shown). The accurate observation of WSCI and E49D models provides a possible explanation for this behaviour; in fact, the H-bond between Glu49 and Arg54 observed in native WSCI, 12 is absent in the mutein model. In E49D, a first possible consequence of the H-bond lack is the increase of the loop flexibility and the adaptability of the inhibitor reactive site to the protease catalytic site, which in turn determines a better enzyme inhibition.…”

“…10,11 As showed by molecular modeling studies, this small protein has a rather compact structure exhibiting an extended solvent-exposed loop (from Val42 to Asp53) containing the inhibitor reactive site P1-P1 0 (Met48-Glu49). 12 The architecture of the loop region, which WSCI shares with other members of the potato inhibitor I family, is characterized by a peculiar conformation depending upon the secondary interactions established between side chains of its residues and those of a small mixed beta-sheet positioned underneath. The degree of flexibility of the region containing the inhibitor reactive site is essential for the productive interaction between this peptide bond and the catalytic site of the target/cognate enzymes.…”

Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves

“…Mass spectra of peptides obtained by chemical or enzymatic digestion were analyzed with a MALDI-TOF micro LR (Waters Micromass Co., Manchester, UK) as reported (Facchiano et al, 2006).…”

Interleukin-10 expression by real-time PCR and homology modelling analysis in the European sea bass (Dicentrarchus Labrax L.)

“…As the sequence identities between the sea bass protein 253 and the human homologous template were lower than 254 30%, we used an accurate procedure to search for the best 255 alignment of sequences, already used and described in our 256 previous works (Facchiano et al, 2001;Marabotti et al, 257 2004;Scapigliati et al, 2004;Costantini et al, 2005; 258 Buonocore et al, 2006;Facchiano et al, 2006) and also in (Thompson et al, 1994) and a few manual refinements were added to account for the position of secondary structures. The program MODELLER (Sali and Blundell, 1993) implemented in the Quanta molecular simulation package (Accelrys, San Diego, CA) was used to build 10 full-atom models by setting 4.0 Angstrom as RMS deviation among the structures of the templates and fully optimized models, with multiple cycles of refinement with conjugate gradient minimization and molecular dynamics with simulated annealing.…”