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Identification and molecular analysis of interaction sites in the MtSEO-F1 protein involved in forisome assembly

: Rose, J.; Visser, F.; Müller, B.; Senft, M.; Groscurth, S.; Sicking, K.F.; Twyman, R.M.; Prüfer, D.; Noll, G.A.

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International journal of biological macromolecules 144 (2020), S.603-614
ISSN: 1879-0003
ISSN: 0141-8130
Zeitschriftenaufsatz, Elektronische Publikation
Fraunhofer IME ()

Forisomes are large mechanoprotein complexes found solely in legumes such as Medicago truncatula. They comprise several “sieve element occlusion by forisome” (SEO-F) subunits, with MtSEO-F1 as the major structure-forming component. SEO-F proteins possess three conserved domains –an N-terminal domain (SEO-NTD), a potential thioredoxin fold, and a C-terminal domain (SEO-CTD)– but structural and biochemical data are scarce and little is known about the contribution of these domains to forisome assembly. To identify key amino acids involved in MtSEO-F1 dimerization and complex formation, we investigated protein-protein interactions by bimolecular fluorescence complementation and the analysis of yeast two-hybrid and random mutagenesis libraries. We identified a SEO-NTD core region as the major dimerization site, with abundant hydrophobic residues and rare charged residues suggesting dimerization is driven by the hydrophobic effect. We also found that ~45% of the full-length MtSEO-F1 sequence must be conserved for higher-order protein assembly, indicating that large interaction surfaces facilitate stable interactions, contributing to the high resilience of forisome bodies. Interestingly, the removal of 62 amino acids from the C-terminus did not disrupt forisome assembly. This is the first study unraveling interaction sites and mechanisms within the MtSEO-F1 protein at the level of dimerization and complex formation.