Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg2+ Transport System

Abstract

Recent crystal structures of the CorA Mg2؉ transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg 2؉ -selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg 2؉ selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.The magnesium ion is essential for life and performs crucial roles in biology. As the major intracellular divalent cation,

Structural insights into the mechanisms of Mg 2+ uptake, transport, and gating by CorA

“…Therefore, the Mg 2+ in the N280-ring is apparently partially hydrated and coordinates with the N280 hydroxyl groups via the water molecules in the first hydration shell. Although the role of the GMN motif had previously been addressed to be involved in ion transport (1,14,15), this is unique structural evidence for the direct role of the Asn of the GMN motif in Mg 2+ uptake. The Asn-ring, because of its geometry, apparently allows the entry of only partially hydrated Mg 2+ .…”

“…G278 creates a hinge that allows the hydrophobic M279 to be kept fixed in a highly hydrophobic environment. A mutation in any of these three residues results in the loss of CorA transport activity (15). Thus, this arrangement of the GMN motif appears to create a perfect entry for partially hydrated Mg 2+ , explaining why this motif has been maintained throughout the evolution of the CorA family and its eukaryotic homologs (1,3).…”

MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces cerevisiae

“…The N-terminal regions of the subunits formed a cytosolic ''funnel'' domain that incorporated several apparent Mg-binding sites, suggesting a regulatory role for this domain. Genetic studies provided evidence that the binding of Mg ions to these sites altered the conformation of the complex and decreased channel activity (Payandeh and Pai 2006;Payandeh et al 2008). The activity of the Mrs2 protein was also shown to be dependent on Mg concentration (Schindl et al 2007), suggesting that both prokaryotic and eukaryotic MIT proteins can respond directly to the cytosolic or matrix Mg concentration to promote homeostasis.…”

“…Mg deficiency might also directly upregulate the activity of the Alr proteins. Although little is known about Alr1 regulation, the activity of bacterial and yeast CorA proteins is coupled to Mg 21 availability via the operation of a Mg-sensing domain located in the cytosol or mitochondrial matrix (Schindl et al 2007;Payandeh et al 2008), and a similar mechanism may operate for the Alr proteins.…”

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

“…Forming the outer wall of the funnel, α5 and α6 contain an unusual amount of negatively charged residues, which confront the basic sphincter (KKKKWL motif of α8 and K292 of α7). Two intracellular Mg 2+ -binding sites (M1 and M2) function as an intracellular divalent cation sensor (DCS) between the intracellular end of α7 and α3′ (residues and distances in protomers B to E are indicated by the addition of prime symbols ′ to ′′′′), where M1 may be the primary regulatory site in TmCorA (21). In a recent computational study (22), removal of the ten regulatory ions from M1 and M2 led to changes in the tilt of α7, resulting in an iris-like dilation of the pore and subsequent hydration of the hydrophobic pore region between M302 and M291 (MM stretch), suggesting an allosteric mechanism coupling intracellular Mg 2+ concentration to hydrophobic gating.…”