Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I.

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure-activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.

Crystal structures of a novel family IV esterase in free and substrate-bound form.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).

Crystal structures of apo and inhibitor-bound TGFßR2 kinase domain: insights into TGFßR isoform selectivity.

The cytokine TGF-ß modulates a number of cellular activities and plays a critical role in development, hemostasis and physiology, as well as in diseases including cancer and fibrosis. TGF-ß signals through two transmembrane serine/threonine kinase receptors: TGFßR1 and TGFßR2. Multiple structures of the TGFßR1 kinase domain are known, but the structure of TGFßR2 remains unreported. Wild-type TGFßR2 kinase domain was refractory to crystallization, leading to the design of two mutated constructs: firstly, a TGFßR1 chimeric protein with seven ATP-site residues mutated to their counterparts in TGFßR2, and secondly, a reduction of surface entropy through mutation of six charged residues on the surface of the TGFßR2 kinase domain to alanines. These yielded apo and inhibitor-bound crystals that diffracted to high resolution (<2 Å). Comparison of these structures with those of TGFßR1 reveal shared ligand contacts as well as differences in the ATP-binding sites, suggesting strategies for the design of pan and selective TGFßR inhibitors.

Two-dimensional crystal structure of aquaporin-4 bound to the inhibitor acetazolamide.

Acetazolamide (AZA) reduces the water permeability of aquaporin-4, the predominant water channel in the brain. We determined the structure of aquaporin-4 in the presence of AZA using electron crystallography. Most of the features of the 5-Å density map were consistent with those of the previously determined atomic model. The map showed a protruding density from near the extracellular pore entrance, which most likely represents the bound AZA. Molecular docking simulations supported the location of the protrusion as the likely AZA-binding site. These findings suggest that AZA reduces water conduction by obstructing the pathway at the extracellular entrance without inducing a large conformational change in the protein.

New crystal structures of adenylate kinase from Streptococcus pneumoniae D39 in two conformations.

Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase from Streptococcus pneumoniae D39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groups P21 and P1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Šresolution, respectively, are reported. The new ligand-free crystal belonged to space group C2, with unit-cell parameters a=73.5, b=54.3, c=62.7 Å, ß=118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cα atoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitor P1,P5-bis(adenosine-5'-)pentaphosphate (Ap5A) belonged to space group P1, with unit-cell parameters a=53.9, b=62.3, c=63.0 Å, α=101.9, ß=112.6, γ=89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation.