Dissecting the conformational complexity and mechanism of a bacterial heme transporter.

Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.

The cryoEM structure of cytochrome bd from C. glutamicum provides novel insights into structural properties of actinobacterial terminal oxidases.

Cytochromes bd are essential for microaerobic respiration of many prokaryotes including a number of human pathogens. These enzymes catalyze the reduction of molecular oxygen to water using quinols as electron donors. Their importance for prokaryotic survival and the absence of eukaryotic homologs make these enzyme ideal targets for antimicrobial drugs. Here, we determined the cryoEM structure of the menaquinol-oxidizing cytochrome bd-type oxygen reductase of the facultative anaerobic Actinobacterium Corynebacterium glutamicum at a resolution of 2.7 Å. The obtained structure adopts the signature pseudosymmetrical heterodimeric architecture of canonical cytochrome bd oxidases formed by the core subunits CydA and CydB. No accessory subunits were identified for this cytochrome bd homolog. The two b-type hemes and the oxygen binding heme d are organized in a triangular geometry with a protein environment around these redox cofactors similar to that of the closely related cytochrome bd from M. tuberculosis. We identified oxygen and a proton conducting channels emerging from the membrane space and the cytoplasm, respectively. Compared to the prototypical enzyme homolog from the E. coli, the most apparent difference is found in the location and size of the proton channel entry site. In canonical cytochrome bd oxidases quinol oxidation occurs at the highly flexible periplasmic Q-loop located in the loop region between TMHs six and seven. An alternative quinol-binding site near heme b 595 was previously identified for cytochrome bd from M. tuberculosis. We discuss the relevance of the two quinol oxidation sites in actinobacterial bd-type oxidases and highlight important differences that may explain functional and electrochemical differences between C. glutamicum and M. tuberculosis. This study expands our current understanding of the structural diversity of actinobacterial and proteobacterial cytochrome bd oxygen reductases and provides deeper insights into the unique structural and functional properties of various cytochrome bd variants from different phylae.

Short-chain aurachin D derivatives are selective inhibitors of E. coli cytochrome bd-I and bd-II oxidases.

Cytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure-activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.

Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

Structural basis for amino acid exchange by a human heteromeric amino acid transporter.

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b[0,+]AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b(0,+)AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid-binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

Optogenetic Control by Pulsed Illumination.

Sensory photoreceptors evoke numerous adaptive responses in nature and serve as light-gated actuators in optogenetics to enable the spatiotemporally precise, reversible, and noninvasive control of cellular events. The output of optogenetic circuits can often be dialed in by varying illumination quality, quantity, and duration. A programmable matrix of light-emitting diodes has been devised to efficiently probe the response of optogenetic systems to intermittently applied light of varying intensity and pulse frequency. Circuits for light-regulated gene expression markedly differed in their responses to pulsed illumination of a single color which sufficed for their sequential triggering. In addition to quantity and quality, the pulse frequency of intermittent light hence provides a further input variable for output control in optogenetics and photobiology. Pulsed illumination schemes allow the reduction of overall light dose and facilitate the multiplexing of several lightdependent actuators and reporters.