Abstract
Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower, Helianthus annuus. The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.
Highlights
Asparaginyl endopeptidases (AEPs) are a group of asparagine/aspartic acid (Asx) specific proteases that have been classified as belonging to the C13 family of cysteine proteases based on the presence of a His-Gly-spacer-Ala-Cys motif (Hara-Nishimura et al, 1993; Chen et al, 1997; Mathieu et al, 2002; Shafee et al, 2015)
Catalytic domain of HaAEP1 purified at pH 4
In order to obtain an active form of a plant AEP, a ~51 kDa pro-HaAEP1 lacking an endoplasmic reticulum signal sequence and including a N-terminal His-tag was expressed in Escherichia coli and purified by nickel affinity chromatography before being activated at pH 4.0 overnight
Summary
Asparaginyl endopeptidases (AEPs) are a group of asparagine/aspartic acid (Asx) specific proteases that have been classified as belonging to the C13 family of cysteine proteases based on the presence of a His-Gly-spacer-Ala-Cys motif (Hara-Nishimura et al, 1993; Chen et al, 1997; Mathieu et al, 2002; Shafee et al, 2015). The recent discovery that evolutionarily distinct plant families have repeatedly recruited AEPs to catalyze the formation of ribosomally synthesized and post-translationally modified peptides (RiPPs), through the macrocyclization of linear precursor sequences, has caught the attention of drug designers keen to overcome the current inefficiencies in native chemical ligation that limit the therapeutic use of cyclic peptides (Pattabiraman and Bode, 2011; Mylne et al, 2012; Arnison et al, 2013). Such therapeutic cyclic peptides are viewed by many to have the potential to capitalize on a niche in the current pharmaceutical market by virtue of their intermediate size
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