Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA.

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

Conformation-specific synthetic antibodies discriminate multiple functional states of the ion channel CorA.

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg 2+ , and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg 2+ . Two sABs from these selections, C12 and C18, showed different degrees of Mg 2+ -sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg 2+ -depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg 2+ -depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg 2+ ] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

The conformational cycle of prestin underlies outer-hair cell electromotility.

The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier1. Knockout or impairment of prestin causes severe hearing loss2-5. Despite the key role of prestin in hearing, the mechanism by which mammalian prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here we determined the structure of dolphin prestin in six distinct states using single-particle cryo-electron microscopy. Our structural and functional data suggest that prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Cl- or SO42-). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of prestin, and inhibits its function by immobilizing prestin in a new conformation. Our data suggest that the bound anion together with its coordinating charged residues and helical dipole act as a dynamic voltage sensor. An analysis of all of the anion-dependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein-membrane interface, suggesting a previously undescribed mechanism of area expansion. Visualization of the electromotility cycle of prestin distinguishes the protein from the closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at a high resolution.

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

CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD ∼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (∼ 10-25 Å) by hinge-like motions as large as ∼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), 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.

Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.

The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αßγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.

1.55†Šresolution X-ray crystal structure of Rv3902c from Mycobacterium tuberculosis.

The crystallographic structure of the Mycobacterium tuberculosis (TB) protein Rv3902c (176 residues; molecular mass of 19.8 kDa) was determined at 1.55 Šresolution. The function of Rv3902c is unknown, although several TB genes involved in bacterial pathogenesis are expressed from the operon containing the Rv3902c gene. The unique structural fold of Rv3902c contains two domains, each consisting of antiparallel ß-sheets and α-helices, creating a hand-like binding motif with a small binding pocket in the palm. Structural homology searches reveal that Rv3902c has an overall structure similar to that of the Salmonella virulence-factor chaperone InvB, with an r.m.s.d. for main-chain atoms of 2.3 Šalong an aligned domain.