Helix-to-Beta-Sheet Transition Drives Self-Assembly of Glutamate Transporter EAA1 Splice Peptides

Truncated isoforms play a critical role in understanding the structural and functional properties of membrane proteins, including glutamate transporters. Here, we molecularly characterize two helical truncated isoforms of the human glutamate transporter EAA1. Using an integrative multi-omics and computational approach, we show that these isoforms, particularly one derived from the N-terminus, do not adopt the canonical transporter fold. Instead, they self-assemble into stable, β-sheet-enriched oligomers, a structure previously unobserved for this protein family. Furthermore, we identified a water-soluble truncated isoform (A0A7P0TAF5) of the membranous canonical EAA1, revealing that self-assembly is not confined to membranous isoforms of EAA1. This finding uncovers a previously unrecognized functional class of truncated isoforms capable of initiating assembly in the soluble state. Our 500ns molecular dynamics simulations further reveal that the N-terminal truncation alters the native conformational dynamics, promoting a transition into semi-helical β-structures over time. In a model bilayer, β-Sheet-driven octamerization of the helical EAA1 isoform A0A7P0Z4F7 induces localized upper leaflet membrane pitting during 250ns all-atom simulation. Helix to β-sheet oligomer transitions is a known pathological hallmark of neurodegenerative disorders such as Alzheimer's disease. Our findings thus uncover a potential new mechanism for glutamate transporter involvement in neurodegeneration and identify the N-terminal domain as a promising therapeutic target. This work highlights how alternative splicing can generate isoforms with novel interaction patterns and distinct molecular conformations.