Structure and inhibition mechanism of the anti-TB drug bedaquiline bound to the ATP synthase rotor from Mycobacteria.

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, ‘coupled’ state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.

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Saccharomyces cerevisiae Chs2 (chitin synthase 2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and is expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during exponential growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS (liquid chromatography tandem MS) experiments on purified Chs2 identify 12 phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division.

We purified the F o complex from the Ilyobacter tartaricus Na + -translocating F 1 F o -ATP synthase and performed a biochemical and structural study. Laser-induced liquid bead ion desorption MS analysis demonstrates that all three subunits of the isolated F o complex were present and in native stoichiometry (ab 2 c 11 ). Cryoelectron microscopy of 2D crystals yielded a projection map at a resolution of 7.0 Å showing electron densities from the c 11 rotor ring and up to seven adjacent helices. A bundle of four helices belongs to the stator a-subunit and is in contact with c 11 . A fifth helix adjacent to the four-helix bundle interacts very closely with a c-subunit helix, which slightly shifts its position toward the ring center. Atomic force microscopy confirms the presence of the F o stator, and a height profile reveals that it protrudes less from the membrane than c 11 . The data limit the dimensions of the subunit a/c-ring interface: Three helices from the stator region are in contact with three c 11 helices. The location and distances of the stator helices impose spatial restrictions on the bacterial F o complex.bioenergetics | membrane protein complex | 2D crystallization | ion translocation mechanism | membrane F o rotor-stator F -type ATP synthases are the major supplier of chemically bound energy in the form of ATP in all living cells. These enzymes use a transmembrane electrochemical ion gradient as an energy source to convert ADP and P i to ATP. Two structurally and functionally distinct domains together form a macromolecular protein complex: the water-soluble F 1 complex (1), with a subunit stoichiometry of α 3 β 3 γδε, and the membraneembedded F o complex (2) (ab 2 c [8][9][10][11][12][13][14][15] ). Both complexes can function as separate and independent rotary motors, fueled by either H + or Na + (F o ) or ATP (F 1 ). Structurally, they are connected by a static peripheral (δb 2 ) and a rotating central (γε) stalk. Ion translocation through the F o complex induces torque, which leads to rotation (3, 4) of the enzyme's rotor (γεc n ). The central stalk γ-subunit rotates inside the F 1 (αβ) 3 headpiece and elicits sequential conformational changes in the three catalytic β-subunits, finally leading to ATP synthesis (5). In the reverse direction, along the same operation principle, the enzyme can act as an ATP hydrolysis-driven ion pump (6, 7).The mechanism of ion translocation and torque generation in the F o complex involves the stator subunits a and b 2 , as well as the rotor ring (c n , c-ring). In bacteria, the outer stalk consists of a pair of b-subunits, each forming a long, membrane-penetrating, and amphiphilic α-helix, which is in contact with the α-, β-, and δ-subunits at the F 1 headpiece of the enzyme, and with the a-subunit and the c-ring in the membrane (8-10). Its coiled and stiff construction was proposed to make it act as a physical barrier against the F o -generated torque (11, 12), but the b-subunit might also be involved in the ion translocation process itself (13,14).Several c-rin...