Structural basis for substrate recognition by the pain-associated neuronal polyamine transporter SLC45A4

Polyamines are essential ubiquitous polycationic molecules involved in diverse cellular processes ranging from gene expression to cell growth and differentiation. The dysregulation of these genes is linked to cancer and neurological disorders. SLC45A4, a recently emerged selective neuronal polyamine transporter, is a critical mediator of polyamine homeostasis and is further linked to pain sensitivity. However, the molecular mechanism underlying substrate recognition and transport remains poorly understood. Here, we present a comprehensive atomistic investigation of SLC45A4 alone and interactions with three major polyamines. We employ knowledge-guided molecular docking and all-atom molecular dynamics simulations in lipid mimetic bilayers at μ-seconds time scale to model the binding modes of spermidine, spermine, and putrescine to SLC45A4. Our results reveal a substrate-dependent landscape in which high-affinity putrescine maintains structural fidelity, whereas long spermine triggers conformational expansions through allosteric decoupling and plug domain unwinding. Furthermore, we identified a conserved cholesterol motif i.e., Leu104, Leu114 and Ala125 which acts as an allosteric splint to stabilize the transporter, demonstrating that a realistic lipid environment is essential to unlock functional dynamics restricted by detergent micelles. Further, the dynamic differences between detergent-solubilized and nanodisc-embedded systems explored herein highlight the micellar cage effect, demonstrating that realistic membrane simulations are essential for capturing MFS alternating-access motions and establishing a structural framework for SLC45A4 drug discovery. These results provide an atomic-level model for polyamine recognition and uptake by SLC45A4, thus provides new avenues for developing new pain therapies.