The bacterial glutamate transporter homolog GltPh, a widely used model for sodium-coupled substrate transport in excitatory amino acid transporters (EAATs), contains three Na + -binding sites (Na1, Na2, and Na3) with distinct roles. Among these, the Na2 site is proposed to complete the fully loaded transporter and stabilize the closed state of the gating hairpin 2 (HP2). Our 2.2 Å cryo-EM structure resolved the details of Na + coordination at this site. However, standard atomistic force fields (CHARMM and AMBER) fail to retain Na + at Na2 in extended molecular dynamics (MD) simulations, leading to ion relocation and dissociation, and conformational artifacts such as HP2 opening. We refined the CHARMM force field with quantum mechanics-derived Lennard-Jones parameters for Na2-coordinating atoms. The improved force field stabilized Na + , prevented artificial conformational changes, and enabled long MD simulations consistent with cryo-EM structures. Validation across different conformational states of GltPh confirmed the fidelity of the refined force field. M311, nearly invariant across Na + -coupled glutamate transporter homologs, coordinates Na via a polarized sulfur atom. M311L and M311A mutants showed Na + displacement (2.4 and 1.7 Å) in MD simulations with the refined force field and cryo-EM structures and reduced computed L-Asp binding stability (5.80 and 7.70 kcal/mol). These mutations altered the steric and electrostatic properties and water organization at the Na2 site. Thus, the refined force field overcomes the key deficiency of the standard model in reproducing Na2 site stability, reveals the basis of M311 functional importance in Na + coupling, and provides a validated computational framework for probing the energetics and kinetics of sodium-dependent transitions in GltPh and EAATs.
Akher et al. (Sun,) studied this question.