Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Background: Glt(Ph) couples uptake of one aspartate to the cotransport of three Na+. Results: Slow binding of Na+ takes place before aspartate binding and has high activation energy. Conclusion: Binding of Na+ is accompanied by a large conformational change enabling subsequent high affinity binding of aspartate. Significance: Insight into the binding kinetics of coupling ions is essential to deduce a mechanism of transport. Glt(Ph) 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 that take up the neurotransmitter glutamate. Each protomer in Glt(Ph) 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 Glt(Ph). Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apoprotein (K-d 120 mm), with a particularly low k(on) value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na+ requires a very high activation energy (E-a 106.8 kJ mol(-1)) and consequently has a large Q(10) 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 K-d for Na+) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k(on) of 1.4 x 10(5) m(-1)s(-1)), with low E-a and Q(10) values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding.