Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor

While the slipknot topology in proteins has been known for over a decade, its evolutionary origin is still a mystery. We have identified a previously overlooked slipknot motif in a family of two-domain membrane transporters. Moreover, we found that these proteins are homologous to several families of unknotted membrane proteins. This allows us to directly investigate the evolution of the slipknot motif. Based on our comprehensive analysis of 17 distantly related protein families, we have found that slipknotted and unknotted proteins share a common structural motif. Furthermore, this motif is conserved on the sequential level as well. Our results suggest that, regardless of topology, the proteins we studied evolved from a common unknotted ancestor single domain protein. Our phylogenetic analysis suggests the presence of at least seven parallel evolutionary scenarios that led to the current diversity of proteins in question. The tools we have developed in the process can now be used to investigate the evolution of other repeated-domain proteins. In proteins with the slipknot topology, the polypeptide chain forms a slipknot-a structure that is not necessarily manifest to a naked eye, but it can be detected using mathematical methods. Slipknots are conserved motifs often found at catalytic sites and are directly involved in molecular transport. Although the first proteins with slipknots were found in 2007, many questions remain unanswered, e.g. how these proteins appeared, or whether the slipknotted proteins evolved from unknotted ones or vice versa. Here we provide the first analysis of homologous slipknotted and unknotted transmembrane proteins in order to elucidate their evolutionary relationship. We show that two-domain slipknotted and unknotted membrane transporters share the same one-domain unknotted protein as an ancestor. The ancestor gene duplicated and underwent various diversification and fusion events during the evolution, which have led to the appearance of a large superfamily of secondary active transporters. The slipknot motif seems to have been created by chance after a fusion of two single domain genes. Therefore, we show here that the slipknotted transporter evolved from an unknotted one-domain protein and that there are at least seven different evolutionary scenarios that gave rise to this large superfamily of transporters.