The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its ...architecture is conserved among vertebrates. Synthesis of triiodothyronine (T
) and thyroxine (T
) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.
Urease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion ...of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.
Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction. The free energy difference of cyclic-nucleotide (cAMP/cGMP) binding/unbinding is translated ...into mechanical work to modulate the open/closed probability of the pore, i.e., gating. Despite the recent advances in structural determination of CNG channels, the conformational changes associated with gating remain unknown. Here we examine the conformational dynamics of a prokaryotic homolog of CNG channels, SthK, using high-speed atomic force microscopy (HS-AFM). HS-AFM of SthK in lipid bilayers shows that the CNBDs undergo dramatic conformational changes during the interconversion between the resting (apo and cGMP) and the activated (cAMP) states: the CNBDs approach the membrane and splay away from the 4-fold channel axis accompanied by a clockwise rotation with respect to the pore domain. We propose that these movements may be converted by the C-linker to pull the pore helices open in an iris diaphragm-like mechanism.
We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3–20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and ...spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics.
Abstract
We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable
Microbacterium
sp. strain BR1 and other Actinomycetes to inactivate ...sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when
Microbacterium
sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of
traA
encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.
KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are ...implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently ...heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.
Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA‐like or ...TagA‐like proteins with a conserved N‐terminal domain and varying C‐terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N‐terminal domain, their roles in T6SS biogenesis are fundamentally different.
Synopsis
Comparative analysis of the bacterial Type VI secretion system TssA/TagA subunits that harbor a conserved N‐terminal domain and varying C‐terminal domains reveals at least three distinct functional classes based on their role in sheath assembly.
First class catalyzes sheath assembly and forms a stable dodecamer at the distal end of polymerizing sheath (T6SS sheath assembly chaperone – TsaC).
Second class localizes at the sheath distal end upon extension across the cell and blocks excessive sheath polymerization (T6SS sheath membrane anchor ‐ TsmA).
Third class localizes to the baseplate and is required for the initiation of sheath formation (T6SS sheath assembly baseplate ‐ TsaB).
Comparative analysis of TssA/TagA subunits of the Type VI secretion system reveals three functional classes with distinct roles in sheath dynamics.
HP1 proteins are major components of heterochromatin, which is generally perceived to be an inert and transcriptionally inactive chromatin structure. Yet, HP1 binding to chromatin is highly dynamic ...and robust silencing of heterochromatic genes can involve RNA processing. Here, we demonstrate by a combination of in vivo and in vitro experiments that the fission yeast HP1Swi6 protein guarantees tight repression of heterochromatic genes through RNA sequestration and degradation. Stimulated by positively charged residues in the hinge region, RNA competes with methylated histone H3K9 for binding to the chromodomain of HP1Swi6. Hence, HP1Swi6 binding to RNA is incompatible with stable heterochromatin association. We propose a model in which an ensemble of HP1Swi6 proteins functions as a heterochromatin-specific checkpoint, capturing and priming heterochromatic RNAs for the RNA degradation machinery. Sustaining a functional checkpoint requires continuous exchange of HP1Swi6 within heterochromatin, which explains the dynamic localization of HP1 proteins on heterochromatin.
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► HP1Swi6 prevents translation and facilitates degradation of heterochromatic mRNAs ► Interaction of HP1Swi6 with RNA mechanistically includes the hinge region, CD and NTD ► Silencing, not heterochromatin integrity, is affected in HP1Swi6 RNA-binding mutants ► Histone tail and RNA binding to HP1Swi6 are competitive processes
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