The CorA family of
proteins plays a housekeeping role in the homeostasis
of divalent metal ions in many bacteria and archaea as well as in
mitochondria of eukaryotes, rendering it an important target to study
the mechanisms of divalent transport and regulation across different
life domains. Despite numerous studies, the mechanistic details of
the channel gating and the transport of the metal ions are still not
entirely understood. Here, we use all-atom and coarse-grained molecular
dynamics simulations combined with
in vitro
experiments
to investigate the influence of divalent cations on the function of
CorA. Simulations reveal pronounced asymmetric movements of monomers
that enable the rotation of the α7 helix and the cytoplasmic
subdomain with the subsequent formation of new interactions and the
opening of the channel. These computational results are functionally
validated using site-directed mutagenesis of the intracellular cytoplasmic
domain residues and biochemical assays. The obtained results infer
a complex network of interactions altering the structure of CorA to
allow gating. Furthermore, we attempt to reconcile the existing gating
hypotheses for CorA to conclude the mechanism of transport of divalent
cations via these proteins.
Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.
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