Structural dynamics of GluK2 kainate receptors in apo and partial agonist bound states.

Kainate receptors (KARs) belong to the family of ionotropic glutamate receptors (iGluRs) and are tetrameric ligand-gated ion channels that regulate neurotransmitter release and excitatory synaptic transmission in the central nervous system. While KARs share overall architectures with other iGluR subfamilies, their dynamics are significantly different from those of other iGluRs. KARs are activated by both full and partial agonists. While there is less efficacy with partial agonists than with full agonists, the detailed mechanism has remained elusive. Here, we used cryo-electron microscopy to determine the structures of homomeric rat GluK2 KARs in the absence of ligands (apo) and in complex with a partial agonist. Intriguingly, the apo state KARs were captured in desensitized conformation. This structure confirms the KAR desensitization prior to activation. Structures of KARs complexed to the partial agonist domoate populate in domoate bound desensitized and non-active/non-desensitized states. These previously unseen intermediate structures highlight the molecular mechanism of partial agonism in KARs. Additionally, we show how N-glycans stabilized the ligand-binding domain dimer via cation/anion binding and modulated receptor gating properties using electrophysiology. Our findings provide vital structural and functional insights into the unique KAR gating mechanisms.

The structural features of Acetobacterium woodii F-ATP synthase reveal the importance of the unique subunit γ-loop in Na+ translocation and ATP synthesis.

The Na+ translocating F1 FO ATP synthase from Acetobacterium woodii shows a subunit stoichiometry of α3 :ß3 :γ:δ:ε:a:b2 :(c2/3 )9 :c1 and reveals an evolutionary path between synthases and pumps involving adaptations in the rotor c-ring, which is composed of F- and vacuolar-type c subunits in a stoichiometry of 9 : 1. This hybrid turbine couples rotation with Na+ translocation in the FO part and rotation of the central stalk subunits γ-ε to drive ATP synthesis in the catalytic α3 :ß3 headpiece. Here, we isolated a highly pure recombinant A. woodii F-ATP synthase and present the first projected structure of this hybrid engine as determined by negative-stain electron microscopy and single-particle analysis. The uniqueness of the A. woodii F-ATP synthase is also reflected by an extra 17 amino acid residues loop (195 TSGKVKITEETKEEKSK211 ) in subunit γ. Deleting the loop-encoding DNA sequence (γΔ195-211 ) and purifying the recombinant F-ATP synthase γΔ195-211 mutant provided a platform to study its effect in enzyme stability and activity. The recombinant F-ATP synthase γΔ195-211 mutant revealed the same subunit composition as the wild-type enzyme and a minor reduction in ATP hydrolysis. When reconstituted into proteoliposomes ATP synthesis and Na+ transport were diminished, demonstrating the importance of the γ195-211 loop in both enzymatic processes. Based on a structural model, a coupling mechanism for this enzyme is proposed, highlighting the role of the γ-loop. Finally, the γ195-211 loop of A. woodii is discussed in comparison with the extra γ-loops of mycobacterial and chloroplasts F-ATP synthases described to be involved in species-specific regulatory mechanisms.

Structure and function of Mycobacterium-specific components of F-ATP synthase subunits α and ε.

The Mycobacterium tuberculosis (Mtb) F1FO-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system αchi3:ß3:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the αchi3:ß3:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε6-121, and Mtε1-120, reconstituted with αchi3:ß3:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.

The NMR solution structure of Mycobacterium tuberculosis F-ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine.

Mycobacterium tuberculosis (Mt) F1 F0 ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) is essential for the viability of growing and nongrowing persister cells of the pathogen. Here, we present the first NMR solution structure of Mtε, revealing an N-terminal ß-barrel domain (NTD) and a C-terminal domain (CTD) composed of a helix-loop-helix with helix 1 and -2 being shorter compared to their counterparts in other bacteria. The C-terminal amino acids are oriented toward the NTD, forming a domain-domain interface between the NTD and CTD. The Mtε structure provides a novel mechanistic model of coupling c-ring- and ε rotation via a patch of hydrophobic residues in the NTD and residues of the CTD to the bottom of the catalytic α3 ß3 -headpiece. To test our model, genome site-directed mutagenesis was employed to introduce amino acid changes in these two parts of the epsilon subunit. Inverted vesicle assays show that these mutations caused an increase in ATP hydrolysis activity and a reduction in ATP synthesis. The structural and enzymatic data are discussed in light of the transition mechanism of a compact and extended state of Mtε, which provides the inhibitory effects of this coupling subunit inside the rotary engine. Finally, the employment of these data with molecular docking shed light into the second binding site of the drug Bedaquiline. DATABASE: Structural data are available in the PDB under the accession number 5YIO.