Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter

Multiscale modeling provides a very powerful means of studying complex biological systems. An important component of this strategy involves coarse-grained (CG) simplifications of regions of the system, which allow effective exploration of complex systems. Here we studied aspects of CG modeling of the human zinc transporter ZnT2. Zinc is an essential trace element with 10% of the proteins in the human proteome capable of zinc binding. Thus, zinc deficiency or impairment of zinc homeostasis disrupt key cellular functions. Mammalian zinc transport proceeds via two transporter families: ZnT and ZIP; however, little is known about the zinc permeation pathway through these transporters. As a step towards this end, we herein undertook comprehensive computational analyses employing multiscale techniques, focusing on the human zinc transporter ZnT2 and its bacterial homologue, YiiP. Energy calculations revealed a favorable pathway for zinc translocation via alternating access. We then identified key residues presumably involved in the passage of zinc ions through ZnT2 and YiiP, and functionally validated their role in zinc transport using site-directed mutagenesis of ZnT2 residues. Finally, we use a CG Monte Carlo simulation approach to sample the transition between the inward-facing and the outward-facing states. We present our structural models of the inward- and outward-facing conformations of ZnT2 as a blueprint prototype of the transporter conformations, including the putative permeation pathway and participating residues. The insights gained from this study may facilitate the delineation of the pathways of other zinc transporters, laying the foundations for the molecular basis underlying ion permeation. This may possibly facilitate the development of therapeutic interventions in pathological states associated with zinc deficiency and other disorders based on loss-of-function mutations in solute carriers.Author summary: Herein we employed multiscale modeling and electrostatic energy calculations to delineate, for the first time, a putative zinc permeation pathway, from the cytoplasm into intracellular vesicles (for ZnT2) or to the extracellular milieu (for YiiP), along the membrane-spanning domain of the human zinc transporter ZnT2 and its E. coli homologue, YiiP. These computational findings were functionally validated using site-directed mutagenesis of ZnT2 residues predicted to reside along the putative zinc permeation pathway and zinc transport assay. Our results shed light on the transport mechanisms of ZnT2 and YiiP and pave the way towards the elucidation of the zinc translocation mechanism in other ZnT family members. Furthermore, these findings could also be harnessed to the possible development of therapeutic interventions in zinc-associated pathologies.