Identification of New Key Players for Ferrous Iron Export in the Asymmetric Inner Gate of Human Ferroportin 1

The Major Facilitator Superfamily (MFS) is the largest known family of secondary transporters. These proteins share a common architecture comprising two lobes, each including 6 transmembrane (TM) helices, related by twofold pseudosymmetry. They transport a wide range of substrates through large conformational changes relying on the opening and closing of gates located on either side of biological membranes. Human ferroportin 1 (HsFPN1), the sole characterized mammalian iron exporter, follows this pattern. It is, however, characterized by an unusual intracellular gate, formed by two asymmetric networks of non-covalent bonds linking the two lobes. We studied the behavior of these networks in all-atom molecular dynamics simulations and functionally assessed the effect of alanine substitutions on HsFPN1 plasma membrane expression and iron export activity. We identified two new critical residues, Arg156 and Tyr318, connecting the networks to each other and to one of two metal-coordinating sites, located in an unwound region of TM7. We extended the analysis to a previously unreported missense variation, p.Gln478Arg, which was found to have a very strong impact on one of the two inter-lobe connection networks, and to result in a significant HsFPN1 loss-of-function. This led us to present the p.Gln478Arg substitution as a new pathogenic variation causing ferroportin disease. Together, our results provide new insights into the structure and dynamics of the human FPN1 inner gate and its asymmetry, shedding light on its potential role in the mechanism of iron export while offering a framework to better understand previously unexplained clinical observations.