Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases.

Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of diverse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the "marker of ligase activity", together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.

Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor.

Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.

SCORE: predicting the core of protein models.

MOTIVATION: The prediction of the regions of homology models that can be 'restrained by' or 'copied from' the basis structures is a vital step in correct model generation, because these regions are the models most accurate part. However, there is no ideal method for the identification of their limits. In most algorithms their length depends on the number of family members and definitions of secondary structure. RESULTS: The algorithm SCORE steps away from the conventional definitions of the core to identify from large numbers of basis structures those regions that can be considered structurally related to a target sequence. The use of phi, psi constraints to accurately pinpoint the regions that are conserved across a family and environmentally constrained substitution tables to extend these regions allows SCORE to rapidly (generally in under 1 s, an order of magnitude faster than methods such as MODELLER) identify and build the core of homology models from the alignments of the target sequence to the basis structures. The SCORE algorithm was used to build 114 model cores. In only two cases was the core size less than 50% of the structure and all the cores built had an RMSD of 3.7 A or less to the target structure.