Comprehensive computational analysis of the molecular mechanism of self-incompatibility in Brassicaceae using improved structure prediction

Abstract

Plants employ self-incompatibility (SI) to promote cross-fertilization. In Brassicaceae, this process is regulated by the complex formation of the pistil determinantSreceptor kinase (SRK) and the pollen determinantS-locus protein 11 (SP11, also known asS-locus cysteine-rich protein, SCR). In our previous study, we demonstrated that only the SRK ectodomain (eSRK) and SP11 pairs derived from the sameShaplotype exhibit high binding free energy based on the crystal structures of two eSRK–SP11 complexes inBrassica rapaS8andS9haplotypes and nine computationally predicted complex models. However, predicting the eSRK–SP11 complex structures for the other 100+ S haplotypes and genera remains difficult because of the SP11 polymorphism in sequence and structure. Although protein structure prediction using AlphaFold2 exhibits considerably high accuracy for most protein monomers and complexes, 46% of predicted SP11 structures we tested showed per-residue mean pLDDT < 75. Here, we demonstrate that the use of curated multiple sequence alignment (MSA) for cysteine-rich proteins significantly improves the model accuracy for SP11 and eSRK–SP11 complexes. Additionally, we calculated the binding free energies of the predicted eSRK–SP11 complexes using molecular dynamics (MD) simulations and observed that some Arabidopsis haplotypes formed a binding mode that was critically different from that ofB. rapaS8andS9. Thus, these computational results provide insights into the haplotype-specific eSRK–SP11 binding modes in Brassicaceae and could be adopted as a general method for obtaining more accurate models for other cysteine-rich proteins.