engleski

Plant seryl-tRNA synthetase and metabolic protein BEN1: identification of interaction surfaces by biophysical methods

Aminoacyl-tRNA synthetases (aaRS) attach appropriate amino acids to cognate tRNAs ensuring efficient protein biosynthesis. Their involvement in diverse celullar functions beyond translation opens broad new perspectives in functional proteomics, especially in the field of plant aaRSs which is fairly uninvestigated. To shed light on non-canonical functions of cytosolic seryl-tRNA synthetase (SerRS) from plant Arabidopsis thaliana we conducted yeast two hybrid screen (Y2H) which revealed metabolic protein BEN1 as a highly promising interacting partner. Interaction was further biophysicaly analyzed in vitro using isothermal calorimetry titration (ITC), pull- down, surface plasmon resonance (SPR) and microscale thermophoresis method (MST). Due to the nature of interaction and sensitivity of applied methods we were not able to retrieve positive results using pull-down assay and ITC, but SPR and MST gave us postive confirmation of interaction and information about dissociation constant (Kd constant obtained using SPR = 1, 03 × 10-6 (± 1, 67 × 10-7) mol dm-3, in good agreement with Kd obtained using MST = 4, 45 × 10-7 (± 1, 40 × 10-7) mol dm-3). To determine interaction surfaces and pinpoint regions responsible for SerRS and BEN1 interaction, truncated variants of both SerRS and BEN1 proteins were prepared and protein interactions were analyzed using MST. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension (with or without (His)6 tag) was similar to Kd of the SerRS:BEN1 complex indicating that SerRS basic C- terminal extension does not have any influence on interaction. Furthermore, isolated N-terminal domain of SerRS did not form the complex with BEN1. Taking into account observed data, we concluded that interaction between SerRS and BEN1 involves the central part of SerRS that contains globular catalytic domain. Determining interaction domain of BEN1 was not possible because of aggregation problems in MST assay when using truncated BEN1 version, so this part of investigation is yet to be done. Knowing precise interaction contacts will allow us to understand and propose functional importance of SerRS:BEN1 assembly in plant organism.

protein-protein interaction ; BEN1 ; plant seryl-tRNA synthetase ; microscale thermophoresis ; surface plasmon resonance

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