Abstract
The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.
Highlights
Pepsin-like aspartic proteases (APs) constitute a family of endopeptidases found in all kingdoms of life [1]
The plantspecific insert (PSI) dimer forms a stable complex regardless of pH Equilibrium molecular dynamics simulations of the dimer complex in acidic and neutral conditions revealed that the PSI dimer is stable regardless of pH, evidenced by low and relatively constant root-mean-square deviation (RMSD) values of the dimer trajectories when fitted to the initial coordinates of the crystal structure (Figure 1)
steered molecular dynamics (SMD) simulations typically use pulling velocities that are orders of magnitude higher than those used in AFM pulling or optical tweezers experiments, resulting in comparatively higher pulling forces
Summary
Pepsin-like aspartic proteases (APs) constitute a family of endopeptidases found in all kingdoms of life [1]. Plant APs are unique in that they frequently contain an extra 100-residue domain inserted in the C-terminal lobe, distinguishing them from their microbial and animal counterparts [3] This extra domain, termed the plantspecific insert (PSI) or plant-specific sequence (PSS) [4,5,6,7], belongs to the saposin-like protein (SAPLIP) family and contains the Saposin B (Sap B) protein domain architecture [8]. Examples of SAPLIP function include sphingolipid degradation and antigen presentation [12], haemolytic activity (Na-SLP-1 and Ac-SLP-1) [13], antimicrobial and cytolytic activity (NK-lysin and granulysin) [14,15] and fusion of large unilamellar anionic vesicles in vitro (Sap C and recombinant PSI expressed without the parent AP) [9,10,11]. Expressed free-form potato PSI has been shown to display potentially useful functionalities in vitro including antimicrobial activity against both plant and human pathogens [16], as well as anticancer activity against leukaemia cells without having lymphocyte toxicity [17]
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