Abstract
Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.
Highlights
Many plant aspartic proteases contain an additional sequence of ϳ100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting
We propose that understanding structure-function relationships involving plantspecific insert (PSI)-membrane interactions may have relevance to non-plant membrane-bound Aspartic proteases (APs) implicated in Alzheimer’s disease beyond the direct elucidation of saposin-like protein (SAPLIP) primary functions
Solanum tuberosum aspartic proteinase (StAP) PSI exhibits a high level of sequence identity (53%) with the SAPLIP domain of prophytepsin PSI (Protein Data Bank code 1QDM; residues 4S–102S), which was used as a model for molecular replacement automated search by PHASER [59]
Summary
Materials—A PSI synthetic gene optimized for expression in Escherichia coli was purchased from Mr Gene GmbH (Regensburg, Germany). Thrombin was added to the dialysates at a 1:2000 mass ratio for incubation at room temperature for at least 12 h followed by reapplication of samples to u-MAC in binding buffer three times consecutively at 2 ml/min to remove the Trx fusion tag. Suspensions of 3 mg/ml PE/PS vesicles were incubated on substrates for 60 min; rinsed with 100 l of 140 mM NaCl, 20 mM MES, pH 4.5, to remove unfused material; and inserted into the fluid cell under 50 l of buffer. After scanning the same region of substrate repeatedly over 30 min, 20 l of 25 M protein solution was injected directly into the buffer in the fluid cell, resulting in 7 M PSI. Three consecutive measurements of five 30-s runs each were averaged using the refractive index for polystyrene, yielding the calculated average sizes and polydispersity indices
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