Kef couples the potassium efflux with proton influx in gram-negative bacteria. The resulting acidification of the cytosol efficiently prevents the killing of the bacteria by reactive electrophilic ...compounds. While other degradation pathways for electrophiles exist, Kef is a short-term response that is crucial for survival. It requires tight regulation since its activation comes with the burden of disturbed homeostasis. Electrophiles, entering the cell, react spontaneously or catalytically with glutathione, which is present at high concentrations in the cytosol. The resulting glutathione conjugates bind to the cytosolic regulatory domain of Kef and trigger activation while the binding of glutathione keeps the system closed. Furthermore, nucleotides can bind to this domain for stabilization or inhibition. The binding of an additional ancillary subunit, called KefF or KefG, to the cytosolic domain is required for full activation. The regulatory domain is termed K
transport-nucleotide binding (KTN) or regulator of potassium conductance (RCK) domain, and it is also found in potassium uptake systems or channels in other oligomeric arrangements. Bacterial RosB-like transporters and K
efflux antiporters (KEA) of plants are homologs of Kef but fulfill different functions. In summary, Kef provides an interesting and well-studied example of a highly regulated bacterial transport system.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus ...contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b
and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
The ability to cope with and adapt to changes in the environment is essential for all organisms. Osmotic pressure is a universal threat when environmental changes result in an imbalance of osmolytes ...inside and outside the cell which causes a deviation from the normal turgor. Cells have developed a potent system to deal with this stress in the form of mechanosensitive ion channels. Channel opening releases solutes from the cell and relieves the stress immediately. In bacteria, these channels directly sense the increased membrane tension caused by the enhanced turgor levels upon hypoosmotic shock. The mechanosensitive channel of small conductance, MscS, from
is one of the most extensively studied examples of mechanically stimulated channels. Different conformational states of this channel were obtained in various detergents and membrane mimetics, highlighting an intimate connection between the channel and its lipidic environment. Associated lipids occupy distinct locations and determine the conformational states of MscS. Not all these features are preserved in the larger MscS-like homologues. Recent structures of homologues from bacteria and plants identify common features and differences. This review discusses the current structural and functional models for MscS opening, as well as the influence of certain membrane characteristics on gating.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae ...in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cytochrome bd quinol:O
oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of ...which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b
is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.
Abstract
Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding ...membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.
The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar ...infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.
Mechanosensitive channels protect bacteria against lysis caused by a sudden drop in osmolarity in their surroundings. Besides the channel of large conductance (MscL) and small conductance (MscS), ...Escherichia coli has five additional paralogs of MscS that are functional and widespread in the bacterial kingdom. Here, we present the structure of YnaI by cryo-electron microscopy to a resolution of 13 Å. While the cytosolic vestibule is structurally similar to that in MscS, additional density is seen in the transmembrane (TM) region consistent with the presence of two additional TM helices predicted for YnaI. The location of this density suggests that the extra TM helices are tilted, which could induce local membrane curvature extending the tension-sensing paddles seen in MscS. Off-center lipid-accessible cavities are seen that resemble gaps between the sensor paddles in MscS. The conservation of the tapered shape and the cavities in YnaI suggest a mechanism similar to that of MscS.
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•First structure of a large member of the MscS family of mechanosensitive channels•The density map indicates extended sensor paddles of tilted transmembrane helices•Lipids access the core of YnaI and fill cavities which are similar to gaps in MscS•These cavities and the tilted helices could lay the basis for tension sensing
Mechanosensitive channels of the MscS family are found in all kingdoms of life and protect bacteria against osmotic shock. Böttcher et al. provide the first structural insights into a larger family member, YnaI. Despite YnaI having additional transmembrane helices, important features are conserved, suggesting a common mechanism.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We describe a mechanosensitive (MS) channel that has mechanosensitive channel of miniconductance (MscM) activity, and displays unique properties with respect to gating. Mechanosensitive channels ...respond to membrane tension, are ubiquitous from bacteria to man, and exhibit a great diversity in structure and function. These channels protect Bacteria and Archaea against hypoosmotic shock and are critical determinants of shape in chloroplasts. Given the dominant roles played in bacteria by the mechanosensitive channel of small conductance (MscS) and the mechanosensitive channel of large conductance (MscL), the role of the multiple MS channel homologs observed in most organisms remains obscure. Here we demonstrate that a MscS homolog, YbdG, extends the range of hypoosmotic shock that Escherichia coli cells can survive, but its expression level is insufficient to protect against severe shocks. Overexpression of the YbdG protein provides complete protection. Transcription and translaiton of the ybdG gene are enhanced by osmotic stress consistent with a role for the protein in survival of hypoosmotic shock. Measurement of the conductance of the native channel by standard patch clamp methods was not possible. However, a fully functional YbdG mutant channel, V229A, exhibits a conductance in membrane patches consistent with MscM activity. We find that MscM activities arise from more than one gene product because ybdG deletion mutants still exhibit an occasional MscM-like conductance. We propose that ybdG encodes a low-abundance MscM-type MS channel, which in cells relieves low levels of membrane tension, obviating the need to activate the major MS channels, MscS and MscL.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Mechanosensitive (MS) channels provide protection against hypo-osmotic shock in bacteria whereas eukaryotic MS channels fulfil a multitude of important functions beside osmoregulation. Interactions ...with the membrane lipids are responsible for the sensing of mechanical force for most known MS channels. It emerged recently that not only prokaryotic, but also eukaryotic, MS channels are able to directly sense the tension in the membrane bilayer without any additional cofactor. If the membrane is solely viewed as a continuous medium with specific anisotropic physical properties, the sensitivity towards tension changes can be explained as result of the hydrophobic coupling between membrane and transmembrane (TM) regions of the channel. The increased cross-sectional area of the MS channel in the active conformation and elastic deformations of the membrane close to the channel have been described as important factors. However, recent studies suggest that molecular interactions of lipids with the channels could play an important role in mechanosensation. Pockets in between TM helices were identified in the MS channel of small conductance (MscS) and YnaI that are filled with lipids. Less lipids are present in the open state of MscS than the closed according to MD simulations. Thus it was suggested that exclusion of lipid fatty acyl chains from these pockets, as a consequence of increased tension, would trigger gating. Similarly, in the eukaryotic MS channel TRAAK it was found that a lipid chain blocks the conducting path in the closed state. The role of these specific lipid interactions in mechanosensation are highlighted in this review.
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