Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis.
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ABSTRACT:
SUBMITTER: Hattori M
PROVIDER: S-EPMC2782099 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Magnesium is an essential ion for numerous physiological processes. MgtE is a Mg2+ selective channel involved in the maintenance of intracellular Mg2+ homeostasis, whose gating is regulated by intracellular Mg2+ levels. Here, we report that ATP binds to MgtE, regulating its Mg2+-dependent gating. Crystal structures of MgtE-ATP complex show that ATP binds to the intracellular CBS domain of MgtE. Functional studies support that ATP binding to MgtE enhances the intracellular domain affinity for Mg2+ within physiological concentrations of this divalent cation, enabling MgtE to function as an in vivo Mg2+ sensor. ATP dissociation from MgtE upregulates Mg2+ influx at both high and low intracellular Mg2+ concentrations. Using site-directed mutagenesis and structure based-electrophysiological and biochemical analyses, we identify key residues and main structural changes involved in the process. This work provides the molecular basis of ATP-dependent modulation of MgtE in Mg2+ homeostasis.MgtE is an Mg2+ transporter involved in Mg2+ homeostasis. Here, the authors report that ATP regulates the Mg+2-dependent gating of MgtE and use X-ray crystallography combined with functional studies to propose the molecular mechanisms involved in this process.
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Project description:Magnesium ions (Mg2+) are divalent cations essential for various cellular functions. Mg2+ homeostasis is maintained through Mg2+ channels such as MgtE, a prokaryotic Mg2+ channel whose gating is regulated by intracellular Mg2+ levels. Our previous crystal structure of MgtE in the Mg2+-bound, closed state revealed the existence of seven crystallographically-independent Mg2+-binding sites, Mg1-Mg7. The role of Mg2+-binding to each site in channel closure remains unknown. Here, we investigated Mg2+-dependent changes in the structure and dynamics of MgtE using nuclear magnetic resonance spectroscopy. Mg2+-titration experiments, using wild-type and mutant forms of MgtE, revealed that the Mg2+ binding sites Mg1, Mg2, Mg3, and Mg6, exhibited cooperativity and a higher affinity for Mg2+, enabling the remaining Mg2+ binding sites, Mg4, Mg5, and Mg7, to play important roles in channel closure. This study revealed the role of each Mg2+-binding site in MgtE gating, underlying the mechanism of cellular Mg2+ homeostasis.
| S-EPMC5882242 | biostudies-literature
Project description:MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and are important participants in the maintenance of cellular Mg2+ homeostasis. The previous high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a recognition mechanism of MgtE for Mg2+ ions. In contrast, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 Å resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ ∼500-fold more strongly than to Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 Å resolution), revealing hexahydrated Ca2+. These results provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.
| S-EPMC9708914 | biostudies-literature
Project description:Magnesium is the most abundant divalent cation present in the cell, and an abnormal Mg2+ homeostasis is associated with several diseases in humans. However, among ion channels, the mechanisms of intracellular regulation and transport of Mg2+ are poorly understood. MgtE is a homodimeric Mg2+-selective channel and is negatively regulated by high intracellular Mg2+ concentration where the cytoplasmic domain of MgtE acts as a Mg2+ sensor. Most of the previous biophysical studies on MgtE have been carried out in detergent micelles and the information regarding gating-related structural dynamics of MgtE in physiologically-relevant membrane environment is scarce. In this work, we monitored the changes in gating-related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in micelles and membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain site-specific information on the gating-related structural dynamics of MgtE in membrane-mimetic systems. Our results indicate that Mg2+-induced gating might involve the possibility of a 'conformational wave' from the cytosolic N-domain to transmembrane domain of MgtE. Although MgtE is responsive to Mg2+-induced gating in both micelles and membranes, the organization and dynamics of MgtE is substantially altered in physiologically important phospholipid membranes compared to micelles. This is accompanied by significant changes in hydration dynamics and conformational heterogeneity. Overall, our results highlight the importance of lipid-protein interactions and are relevant for understanding gating mechanism of magnesium channels in general, and MgtE in particular.
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Project description:YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670-26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19-->Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET(+), thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an approximately 800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19-->Cys MscL in the presence of YggB, but a downshock of only approximately 400 mmol/kg is required in the absence of YggB. In parallel, Leu-19-->Cys MscL, stretched open by large sustained suction in the presence of MTSET(+) in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.
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Project description:Here we explore the mechanism and associated structure-function implications of loss of function for epithelial Na(+) channel (ENaC) containing a pseudohypoaldosteronism type 1 (PHA-1)-causing missense point mutation. As expected, human ENaC that contained subunits harboring PHA-1-causing substitutions within an absolutely conserved, cytosolic Gly residue (e.g., βG37S) had significantly less activity. Unexpectedly, though, such substitution also results in voltage sensitivity with greater activity at hyperpolarizing potentials. This is a consequence of voltage-dependent changes in the single-channel open probability and is not species- or subunit-dependent. Voltage sensitivity in PHA-1 mutants stems from the disruption of critical structure, rather than the development of new properties resulting from the introduction of novel side chains. Residues near the conserved His-Gly sequence of G95 in α-mENaC are particularly important for voltage sensing. Although substitution of I93 in α-mENaC results in voltage sensing, it also slows the activation and deactivation kinetics enough to enable capture of the dynamic changes in single-channel open probability that account for changes in macroscopic activity. This provides definitive proof of the mechanism that underlies loss of function. In addition, the voltage dependence of ENaC with PHA-1 substitutions is akin to that which results from substitution of a critical, interfacial Trp residue conserved at the intracellular base of TM1 (e.g., W112 in α-mENaC). Dynamic interactions between similarly positioned His and Trp residues are essential for gating and the girdle-like structure that lines the intracellular mouth of the M2 proton channel. The similar residues in ENaC may serve a shared function, suggesting the possibility of an intracellular girdle just below the mouth of the ENaC pore.
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Project description:We outline several principles that we believe define the gating of two bacterial mechanosensitive channels, MscL and MscS. Serving as turgor regulators in bacteria and other walled cells, these molecules are tangible models for studying conformational transitions in membrane proteins driven directly by membrane tension. MscL, a compact pentamer, reversibly opens a gigantic 30-A pore at near-lytic tensions. MscS, a heptameric complex, exhibits transient activation of a smaller pore at moderate tensions, thereby entering a tension-insensitive inactivated state. By comparing the structures and predicted transitions in these channels, we concluded that opening is commonly achieved through tilting and outward motion of the pore-lining helices, which is kinetically limited by hydration of the pore. The intricate adaptive behavior in MscS appears to depend on specific interhelical associations and the flexibility of the pore-lining helices. We discuss physical factors that may direct the transitions and stabilize main functional states in these channels.
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