Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

Ferlins are complex, multi-domain proteins, involved in membrane trafficking, membrane repair, and exocytosis. The large size of ferlin proteins and the lack of consensus regarding domain boundaries have slowed progress in understanding molecular-level details of ferlin protein structure and function. However, in silico protein folding techniques have significantly enhanced our understanding of the complex ferlin family domain structure. We used RoseTTAFold to assemble full-length models for the six human ferlin proteins (dysferlin, myoferlin, otoferlin, Fer1L4, Fer1L5, and Fer1L6). Our full-length ferlin models were used to obtain objective domain boundaries, and these boundaries were supported by AlphaFold2 predictions. Despite the differences in amino acid sequence between the ferlin proteins, the domain ranges and distinct subdomains in the ferlin domains are remarkably consistent. Further, the RoseTTAFold/AlphaFold2 in silico boundary predictions allowed us to describe and characterize a previously unknown C2 domain, ubiquitous in all human ferlins, which we refer to as C2-FerA. At present, the ferlin domain-domain interactions implied by the full-length in silico models are predicted to have a low accuracy; however, the use of RoseTTAFold and AlphaFold2 as a domain finder has proven to be a powerful research tool for understanding ferlin structure.