Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating

Abstract

Summary CorA, the major Mg2+ uptake system in prokaryotes, is gated by intracellular Mg2+ (KD ~1–2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg2+-bound and Mg2+-free conditions, but EPR spectroscopic studies reveal large Mg2+-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg2+-bound “closed” conformation and in two “open” Mg2+-free states at resolutions of 3.8 A, 7.1 A and 7.1 A, respectively. In the absence of bound Mg2+, four of the five subunits are displaced to variable extents (~10 to ~25 A) by hinge-like motions at the stalk helix as large as ~35°. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg2+, open, asymmetric conformational states, is thus the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery

“…Large and relatively stable complexes such as ribosomes also proved especially amenable to analysis using cryo-EM methods, first at medium resolution (Matadeen et al, 1999; Rawat et al, 2003) and more recently at near-atomic resolution (Amunts et al, 2014; Fischer et al, 2015; Jomaa et al, 2016; Wong et al, 2014). These successes have now been extended to a wide spectrum of protein complexes, including several integral membrane proteins (Bai et al, 2015b; Du et al, 2015; Liao et al, 2013; Matthies et al, 2016). Structures determined by cryo-EM can now reach resolutions as high as 2.2 Å and 2.3 Å, as exemplified by structures of the 465 kDa β-galactosidase (Bartesaghi et al, 2015) and the 540 kDa AAA ATPase p97 (Banerjee et al, 2016).…”

Cryo-EM: beyond the microscope

“…Nanodiscs are essentially small discs of lipid bilayers bounded by a stable protein ring that is thought to form a belt around the periphery of the disc (Figure 2b). Variations in the size of the nanodisc can potentially preclude achieving high resolution structures [57] (Figure 2c), driving the need for improved methods to generate more homogeneously-sized nanodisc assemblies. Although there are only a few membrane proteins whose structures have been determined to high resolution in the nanodisc-embedded state [58,59] (Figure 2d), the fact that structures determined using this approach can also provide information on boundary lipids makes this an appealing alternative to be developed further.…”

“…(c) Three 3D classes of the membrane channel CorA in nanodiscs, showing that nanodisc size can vary significantly, precluding high resolution structure determination by 3D averaging. From [57]. (d) Side (left) and top (right) 2D classes of the membrane protein TRPV1 in nanodiscs.…”

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

“…Besides nowadays various structures a plethora of EPR studies has been performed with regard to conformational changes in the selectivity filter [65,66], the cytoplasmic helical bundle [67,68] and the communication between both features [69]. Further excellent examples for the combined use of structural biology and EPR spectroscopy are present for all classes of ion channels, e.g., for the intracellular Mg 2+ -gated Mg 2+ channel CorA [70][71][72], the nucleotide-and Na + -gated K + channel KtrAB [73], the mechanosensitive channel MscL [74,75], …”

“…Besides nowadays various structures a plethora of EPR studies has been performed with regard to conformational changes in the selectivity filter [65,66], the cytoplasmic helical bundle [67,68] and the communication between both features [69]. Further excellent examples for the combined use of structural biology and EPR spectroscopy are present for all classes of ion channels, e.g., for the intracellular Mg 2+ -gated Mg 2+ channel CorA [70][71][72], the nucleotide-and Na + -gated K + channel KtrAB [73], the mechanosensitive channel MscL [74,75], the cyclic AMP-gated HCN channel [45,76], the voltage-gated Na + channel NavMs [77] and the pentameric receptor channels GLIC [78]. In all cases EPR measurements have been applied to validate existing protein structures, determine the structure of missing regions or distorted elements in structures, or add valuable information of the dynamics of the studied systems to provide a model of activation and regulation.…”