Nucleotide-dependent mechanism of Get3 as elucidated from free energy calculations

The unique topology of tail-anchored (TA) proteins precludes them from utilizing the well-studied cotranslational translocation mechanism of most transmembrane proteins, forcing them into a distinct, posttranslational pathway. In yeast, this process is the guided entry of TA-proteins (GET) pathway, which utilizes a combination of cytosolic and transmembrane proteins to identify a TA protein, transfer it, and insert it into the endoplasmic reticulum membrane. At the center of this mechanism is the Get3 homodimer, which transfers a TA protein between the two GET phases by leveraging energy gained in ATP binding and hydrolysis to undergo significant structural changes from "open" to "closed" conformations. We present all-atom molecular dynamics simulations of Get3 in multiple nucleotide states, and through rigorous potential of mean force calculations, compute the free energy landscape of the Get3 opening/closing pathway. Results agree well with experiments on the nucleotide bias of Get3 open and closed structures in the crystallographically observed no-nucleotide, two ATP, and two ADP states, and also reveal their populations in the asymmetric one ATP and one ADP cases. Structures also compare well with the recently observed "semiopen" conformation and suggest that Get3 may sample this state free in solution and not just when bound to Get1, as observed in experiments. Finally, we present evidence for a unique, "wide-open" conformation of Get3. These calculations describe the nucleotide-dependent thermodynamics of Get3 in solution, and improve our understanding of its mechanism in each phase of the GET cycle.