Structural Dynamics and Residue-Specific Interactions Govern Ammonia Transport in AMT/MEP Transporter Variants

The AMT/MEP/Rh superfamily plays a key role in nitrogen homeostasis. However, the molecular mechanisms governing transport and responses to varying concentration of ammonium remain unclear. Herein, we performed molecular dynamics in order to investigate ammonia (NH₃) transport mechanisms across different structural variants of the AMT transporter protein, including MEP (control), AMT-HK fusion, truncated AMT (HK domain removed), and a functional AMT variant. Structural stability analysis via RMSD and RMSF revealed that the AMT-HK fusion exhibited higher flexibility (~6 Å RMSD) compared to MEP (~1.2 Å), suggesting ammonia-induced conformational changes that may facilitate signal transduction to the HK domain. Truncated AMT showed reduced structural fluctuations (~2 Å RMSD), supporting ammonia's role in modulating dynamics. Transport analysis indicated that MEP facilitated rapid unidirectional NH₃ translocation, while AMT-HK exhibited slower, regulated transport with prolonged NH₃ retention. Intriguingly, truncated AMT displayed partial bidirectional flux, implicating the HK domain in enforcing directionality. Residue interaction mapping revealed distinct NH₃ coordination environments: AMT-HK relied on hydrophobic residues (e.g., ALA322, VAL102) for retention, whereas truncated AMT shifted toward polar residues (e.g., ALA279, GLY333) near the cytoplasmic region. Comparative analysis highlighted key mechanistic differences—MEP utilized histidine-mediated NH₃ transport, while AMT variants depended on phenylalanine clusters (e.g., PHE21, PHE27), with enhanced interactions compensating for HK domain removal. These findings elucidate how structural flexibility, residue chemistry, and domain fusion collectively regulate NH₃ transport efficiency and directionality. The study provides molecular-level insights into AMT transporter function, emphasizing the interplay between conformational dynamics and substrate passage, and establishes a framework for future mechanistic studies.