Bioinformatics classification of the MgtE Mg2+ channel and de novo protein design for the stabilization of its novel subclass

Abstract

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 Thermus thermophilus 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 de novo proteins and determine their crystal structures. A selected de novo protein is fused to a mini-N-type MgtE, enabling successful purification and preliminary cryo-EM imaging. Our findings demonstrate that de novo-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.

1. Introduction

MgtE represents a widely conserved Mg2+ channel in bacteria that plays a critical role in Mg2+ homeostasis [1–4] and contributes to bacterial survival under antibiotic exposure by counteracting hyperpolarization [5]. In Pseudomonas aeruginosa, MgtE inhibits the transcription of the type III secretion system, a critical virulence factor, by promoting the expression of the translation-inhibiting sRNAs RsmY and RsmZ [6,7]. In humans, MgtE homologs are referred to as SLC41A1 to SLC41A3 and have been implicated in various physiological functions and diseases, including Parkinson’s disease [8–11]. Accordingly, the MgtE/SLC41 family of proteins has attracted considerable interest as potential therapeutic targets.

To date, extensive structural and mechanistic studies have been conducted on MgtE [12–14]. These studies revealed that MgtE functions as a “Mg2+-gated Mg2+ channel” where the binding of Mg2+ to the cytoplasmic domain leads to channel inactivation [3,15–17]. High-resolution structural analyses of the MgtE transmembrane (TM) domain have elucidated the mechanism of Mg2+ selectivity [18,19], whereas the cryo-EM structure of MgtE in the absence of the Mg2+ domain has provided insights into the gating mechanism [20]. However, these studies have focused predominantly on Thermus thermophilus MgtE (TtMgtE), and structural and biochemical data for MgtE homologs from other species are extremely limited [21–23]. As a result, the structural and functional diversity of the broadly conserved MgtE family remains largely unexplored. In parallel, the recent explosion in genomic data has made it increasingly feasible to investigate the diversity of specific protein families via genome mining approaches [24,25].

One major challenge in studying membrane proteins such as MgtE lies in their generally low expression levels and poor stability in detergents [26], which significantly hinders structural and functional analyses. Recently, AI-based de novo protein design technologies, such as ProteinMPNN [27] and RF diffusion [28], have rapidly advanced [29] and are expected to facilitate research on membrane proteins [30]. De novo-designed proteins typically exhibit high stability, and fusing them to target membrane proteins may increase their expression and stability, potentially enabling the preparation of large quantities of stable membrane protein samples. In this study, we conducted a bioinformatics analysis of the MgtE family and identified a novel subclass of MgtE proteins with unique domain architectures. Despite extensive expression screening, these proteins could not be stably purified. To overcome this challenge, we designed de novo proteins and successfully determined the crystal structures of several designed proteins. Furthermore, by generating fusion constructs with the de novo-designed protein and the novel MgtE subclass, we achieved successful expression and purification. Overall, this study highlights a promising strategy for stabilizing and purifying membrane proteins via a de novo-designed protein fusion approach.