Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert

Xiaoli Zhao,Jenny (Jingxin) Tian,Hua Yu,Brian C Bryksa,John H Dupuis,Xiuyuan Ou,Zhaohui Qian,Chen Song,Shenlin Wang,Rickey Y Yada

doi:10.1074/jbc.ra120.014311

Abstract

In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.

Highlights

Unlike mammals, plants do not have mobile immune cells and have evolved several natural defense mechanisms in response to pathogen attack [1,2,3,4]
The plant-specific insert (PSI) protein belongs to a large group of proteins—the saposin-like protein (SAPLIP) family [13]
An integrative approach using NMR, FRET, and in silico methods demonstrated the details of the mechanisms of PSI

Summary

Results

Mode B binding (orientation angle ;63°) was observed in all of showed that the PSI dimer can spontaneously bind to the surfa- the four simulations (Fig. 5C). The existence of the unstable interface between the PSI monomer and bilayer, together with the relatively even distribution of the orientation angle of the monomer, suggested that the PSI monomer is probably not as effective as the dimer in bringing two lipid bilayers together and inducing membrane fusion. The combination of our in silico and experimental results led to the proposal of a potential binding pathway: (i) the PSI dimer is able to bind to two nearby vesicles by favorable interactions with their membrane surfaces, whereas the PSI monomer only binds stably with one vesicle on its membrane surface; (ii) the PSI formed a bridge between them via Mode B binding, thereby tethering them together in close proximity, and (iii) through various intermolecular and external forces, the vesicles are pulled even closer by altering the orientation to the metastable Mode A, which may eventually facilitate membrane fusion. The results showed that the interacting residues were almost identical for Mode A in one lipid bilayer and Mode A in the post-fusion state (Fig. S7). The PSI dimer has hydrophobic interfaces on both sites, each of which can bind strongly with the one of the two vesicles, forming a bridge between the two vesicles

Discussion

Experimental procedures