Low affinity and slow Na+-binding precedes high affinity aspartate binding in GltPh

GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters (EAATs) that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions, and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na+ and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apo-protein (Kd 120 mM), with a particularly low kon value (5 M-1s-1). At least two Na+ ions bind prior to aspartate. The binding of Na+ requires very high activation energy (Ea 106.8 kJmol-1) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na+ concentration present. Binding of aspartate was not observed in the absence of Na+, whereas in the presence of high Na+ concentrations (above the Kd for Na+) the dissociation constants for aspartate were in the nanomolar range and the aspartate binding was fast (kon of 1.4*105 M-1s-1), with low Ea and Q10 values (42.6 KJmol-1 and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding