Bioinformatics classification of the MgtE Mg<sup>2</sup><sup>+</sup> channel and de novo protein design for the stabilization of its novel subclass

<p indent="0mm">MgtE channels play crucial roles in Mg²⁺ homeostasis and are implicated in bacterial survival under antibiotic exposure. Previous structural and biophysical studies have focused predominantly on <italic>Thermus thermophilus</italic> MgtE, leaving the structural and mechanistic diversity of MgtE family proteins largely unexplored. In this study, via a genome mining approach, we identify diverse MgtE homologs, including a novel subclass termed the “mini-N type,” which lacks the canonical cytoplasmic N and CBS domains but possesses a unique small N-like domain. Despite extensive expression screening, mini-N-type homologs cannot be stably purified. To address this issue, we design a series of <italic>de novo</italic> proteins and determine their crystal structures. A selected <italic>de novo</italic> protein is fused to a mini-N-type MgtE, enabling successful purification and preliminary cryo-EM imaging. Our findings demonstrate that <italic>de novo</italic>-designed protein fusions serve as powerful tools for stabilizing and purifying otherwise unstable membrane proteins, opening new avenues for structural and functional studies of otherwise inaccessible membrane proteins.