Evolutionary and structural analysis of procirsin, a typical plant aspartic proteinase zymogen

DANIELA LUFRANO; SANDRA E. VAIRO CAVALLI; GUSTAVO PARISI

Oro Verde

Congreso; 3er Congreso Argentino de Bioinformática y Biología Computacional; 2012

Asociación Argentina de Bioinformática y Biología Computacional

In plants, aspartic proteases (APs, EC. 3.4.23) appear to be the second-largest class of proteases being A1 family the best studied and the largest group, classified in these organisms into typical, nucellin-like and atypical proteases [1]. Typical plant APs are synthesized as single-chain preproenzymes characterized by the presence of the plant specific insert (PSI) domain of approximately 100 amino acids, absent in APs from other sources (viruses, bacteria, yeast, fungi and animals). The preproenzymes are subsequently processed into single- or two-chain mature forms where PSI domain is removed. The prosegment and the first residues of the N-terminal portion of the AP precursors have been described to play a critical role in blocking catalytic aspartates and thus preventing autoactivation. Particularly, residue Arg 7 of the propeptide in barley´s typical AP precursor (prophytepsin) is reported to form an ionic interaction with Glu 171 and Asp 178 in mature protein, and together with other hydrophobic and hydrogen bonds, links the propeptide in a way that the Lys 11 and Tyr 13 of the N-terminal region interact with the active site inhibiting the activity of propythepsin. However, the precursor of a typical AP from flowers of Cirsium vulgare (Savi) Ten. (Asteraceae), called procirsin and obtained by heterologus expression, was shown to be active at acidic pH [2]. In order to find possible differences that explain recombinant procirsin activity, we performed a phylogenetic analysis of procirsin and a structural model using Modeller program, further evaluated with the DOPE potential and Prosa II server (score -8.5). We also estimated the variation of the net charge of the propeptides of procirsin and prophythepsin as a pH function. Our analysis shows that procirsin shares a cluster with APs from diverse organisms in which the closest homologous is cyprosin from C. cardunculus (98% of sequence similarity). According to the structural model and the evolutionary analysis, all the residues described as important for biological function, as well as Arg 7p, Lys 11 and Tyr 13 are conserved in procirsin in comparison with all the sequences of the cluster. The large positive charge at acidic pH predicted for the prosegment of procirsin when compared with prophytepsin, could alter the correct localization of the propeptide avoiding the interaction of Lys/Tyr with the catalytic residues and turning the procirsin active at low pH. We propose that as pH increases, the charge in the prosegment decreases allowing the correct conformation to inhibit the proenzyme.