Jun Liao

Structural Insight into the Ion-Exchange Mechanism of the Sodium/Calcium Exchanger

Maintaining Equilibrium Na+/Ca2+ exchangers (NCX) are membrane transporters that maintain the homeostasis of cytosolic Ca2+ and play an essential role in Ca2+ signaling. Despite a long history of physiological work and a large body of functional data, the structural basis underlying the ion exchange mechanism of NCX is poorly understood. Liao et al. (p. 686; see the Perspective by Abramson et al.) present a high-resolution crystal structure of an NCX from Methanococcus jannaschii and demonstrate that this archaeal NCX catalyzes Na+/Ca2+-exchange reactions similar to its eukaryotic counterpart. The structure clarifies the mechanism of ion exchange proteins and reveals the basis for the stoichiometry, cooperativity, and bidirectionality of the reaction. The arrangement of four central ion-binding sites with differing specificities suggests how a membrane transporter protein works. Sodium/calcium (Na+/Ca2+) exchangers (NCX) are membrane transporters that play an essential role in maintaining the homeostasis of cytosolic Ca2+ for cell signaling. We demonstrated the Na+/Ca2+-exchange function of an NCX from Methanococcus jannaschii (NCX_Mj) and report its 1.9 angstrom crystal structure in an outward-facing conformation. Containing 10 transmembrane helices, the two halves of NCX_Mj share a similar structure with opposite orientation. Four ion-binding sites cluster at the center of the protein: one specific for Ca2+ and three that likely bind Na+. Two passageways allow for Na+ and Ca2+ access to the central ion-binding sites from the extracellular side. Based on the symmetry of NCX_Mj and its ability to catalyze bidirectional ion-exchange reactions, we propose a structure model for the inward-facing NCX_Mj.

Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator

ABSTRACT Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment. IMPORTANCE Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan LuxR-type receptors and do not produce AHLs, sensing them from other bacteria. We solved three structures of the E. coli SdiA orphan, in the presence and absence of AHL. SdiA with no AHL is not in an apo state but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL). OCL is ubiquitously found in prokaryotes and eukaryotes and is a phospholipid precursor for membrane biogenesis and a signaling molecule. While AHL renders to SdiA higher stability and DNA-binding affinity, OCL functions as a chemical chaperone placeholder, stabilizing SdiA and allowing for basal activity. Our studies provide crucial mechanistic insights into the ligand regulation of SdiA activity. Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan LuxR-type receptors and do not produce AHLs, sensing them from other bacteria. We solved three structures of the E. coli SdiA orphan, in the presence and absence of AHL. SdiA with no AHL is not in an apo state but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL). OCL is ubiquitously found in prokaryotes and eukaryotes and is a phospholipid precursor for membrane biogenesis and a signaling molecule. While AHL renders to SdiA higher stability and DNA-binding affinity, OCL functions as a chemical chaperone placeholder, stabilizing SdiA and allowing for basal activity. Our studies provide crucial mechanistic insights into the ligand regulation of SdiA activity.

Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

Na+/Ca2+ exchangers use the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+ and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1 Na+/Ca2+-exchange stoichiometry and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion occupancy, thereby explaining the emergence of strictly coupled Na+/Ca2+ antiport.

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5′-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.

The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways

The Na+-Ca2+ exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca2+ homeostasis by moving Ca2+ out of the cell using the electrochemical gradient of Na+ as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na+ and Ca2+ ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.

Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology

NCX_Mj is a sodium–calcium exchanger from the archaebacterium Methanococcus jannaschii, whose crystal structure has been solved. Barthmes et al. use solid supported membrane–based electrophysiology to characterize NCX_Mj and reveal its functional similarity to eukaryotic isoforms.

Succinate-acetate permease from Citrobacter koseri is an anion channel that unidirectionally translocates acetate

Acetate is an important metabolite in metabolism and cell signaling. Succinate-Acetate Permease (SatP) superfamily proteins are known to be responsible for acetate transport across membranes, but the nature of this transport remains unknown. Here, we show that the SatP homolog from Citrobacter koseri (SatP_Ck) is an anion channel that can unidirectionally translocate acetate at rates of the order of ~107 ions/s. Crystal structures of SatP_Ck in complex with multiple acetates at 1.8 Å reveal that the acetate pathway consists of four acetate-binding sites aligned in a single file that are interrupted by three hydrophobic constrictions. The bound acetates at the four sites are each orientated differently. The acetate at the cytoplasmic vestibule is partially dehydrated, whereas those in the main pore body are fully dehydrated. Aromatic residues within the substrate pathway may coordinate translocation of acetates via anion-π interactions. SatP_Ck reveals a new type of selective anion channel and provides a structural and functional template for understanding organic anion transport.