Cryo-EM structures of LolCDE reveal the molecular mechanism of bacterial lipoprotein sorting in Escherichia coli.

Bacterial lipoproteins perform a diverse array of functions including bacterial envelope biogenesis and microbe-host interactions. Lipoproteins in gram-negative bacteria are sorted to the outer membrane (OM) via the localization of lipoproteins (Lol) export pathway. The ATP-binding cassette (ABC) transporter LolCDE initiates the Lol pathway by selectively extracting and transporting lipoproteins for trafficking. Here, we report cryo-EM structures of LolCDE in apo, lipoprotein-bound, and AMPPNP-bound states at a resolution of 3.5 to 4.2 Å. Structure-based disulfide crosslinking, photo-crosslinking, and functional complementation assay verify the apo-state structure and reveal the molecular details regarding substrate selectivity and substrate entry route. Our studies snapshot 3 functional states of LolCDE in a transport cycle, providing deep insights into the mechanisms that underlie LolCDE-mediated lipoprotein sorting in E. coli.

Cryo-EM structure of the nonameric CsgG-CsgF complex and its implications for controlling curli biogenesis in Enterobacteriaceae.

Curli play critical roles in biofilm formation, host cell adhesion, and colonization of inert surfaces in many Enterobacteriaceae. In Escherichia coli, curli biogenesis requires 7 curli-specific gene (csg) products-CsgA through G-working in concert. Of them, CsgG and CsgF are 2 outer membrane (OM)-localized components that consists of the core apparatus for secretion and assembly of curli structural subunits, CsgB and CsgA. Here, we report the cryogenic electron microscopy (cryo-EM) structure of CsgG in complex with CsgF from E. coli. The structure reveals that CsgF forms a stable complex with CsgG via a 1:1 stoichiometry by lining the upper lumen of the nonameric CsgG channel via its N-terminal 27 residues, forming a funnel-like entity plugged in the CsgG channel and creating a unique secretion channel with 2 constriction regions, consistent with the recently reported structure of the CsgG-CsgF complex. Functional studies indicate that export of CsgF to the cell surface requires the CsgG channel, and CsgF not only functions as an adaptor that bridges CsgB with CsgG but also may play important roles in controlling the rates of translocation and/or polymerization for curli structural subunits. Importantly, we found that a series of CsgF-derived peptides are able to efficiently inhibit curli production to E. coli when administrated exogenously, highlighting a potential strategy to interfere biofilm formation in E. coli strains.

Effect of charge on protein preferred orientation at the air-water interface in cryo-electron microscopy.

The air-water interface (AWI) tends to adsorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, some of the nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. Our results indicate that proteins adsorb to charged interface and the negative charge of the AWI plays an important role in adsorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed by adding anionic detergent at a concentration between 0.002% and 0.005%. Using this method, the protein particles exhibited a more evenly distributed orientations and still adsorbed to the AWI enabling them embedding in a thin layer of ice with high concentration, which will benefit the cryo-EM structural determination.

Cryo-EM structures of LptB2FG and LptB2FGC from Klebsiella pneumoniae in complex with lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most Gram-negative bacteria. LPS transport from the inner membrane (IM) to the OM is achieved by seven lipopolysaccharide transport proteins (LptA-G). LptB2FG, an type VI ATP-binding cassette (ABC) transporter, forms a stable complex with LptC, extracts LPS from the IM and powers LPS transport to the OM. Here we report the cryo-EM structures of LptB2FG and LptB2FGC from Klebsiella pneumoniae in complex with LPS. The KpLptB2FG-LPS structure provides detailed interactions between LPS and the transporter, while the KpLptB2FGC-LPS structure may represent an intermediate state that the transmembrane helix of LptC has not been fully inserted into the transmembrane domains of LptB2FG.

Distinct Perception Mechanisms of BACH1 Quaternary Structure Degrons by Two F-box Proteins under Oxidative Stress.

The transcription factor BACH1 regulates heme homeostasis and oxidative stress responses and promotes cancer metastasis upon aberrant accumulation. Its stability is controlled by two F-box protein ubiquitin ligases, FBXO22 and FBXL17. Here we show that the homodimeric BTB domain of BACH1 functions as a previously undescribed quaternary structure degron, which is deciphered by the two F-box proteins via distinct mechanisms. After BACH1 is released from chromatin by heme, FBXO22 asymmetrically recognizes a cross-protomer interface of the intact BACH1 BTB dimer, which is otherwise masked by the co-repressor NCOR1. If the BACH1 BTB dimer escapes the surveillance by FBXO22 due to oxidative modifications, its quaternary structure integrity is probed by a pair of FBXL17, which simultaneously engage and remodel the two BTB protomers into E3-bound monomers for ubiquitination. By unveiling the multifaceted regulatory mechanisms of BACH1 stability, our studies highlight the abilities of ubiquitin ligases to decode high-order protein assemblies and reveal therapeutic opportunities to block cancer invasion via compound-induced BACH1 destabilization.

Addressing compressive deformation of proteins embedded in crystalline ice.

For cryoelectron microscopy (cryo-EM), high cooling rates have been required for preparation of protein samples to vitrify the surrounding water and avoid formation of damaging crystalline ice. Whether and how crystalline ice affects single-particle cryo-EM is still unclear. Here, single-particle cryo-EM was used to analyze three-dimensional structures of various proteins and viruses embedded in crystalline ice formed at various cooling rates. Low cooling rates led to shrinkage deformation and density distortions on samples having loose structures. Higher cooling rates reduced deformations. Deformation-free proteins in crystalline ice were obtained by modifying the freezing conditions, and reconstructions from these samples revealed a marked improvement over vitreous ice. This procedure also increased the efficiency of cryo-EM structure determinations and was essential for high-resolution reconstructions.

An electron counting algorithm improves imaging of proteins with low-acceleration-voltage cryo-electron microscope.

Relative to the 300-kV accelerating field, electrons accelerated under lower voltages are potentially scattered more strongly. Lowering the accelerate voltage has been suggested to enhance the signal-to-noise ratio (SNR) of cryo-electron microscopy (cryo-EM) images of small-molecular-weight proteins (<100 kD). However, the detection efficient of current Direct Detection Devices (DDDs) and temporal coherence of cryo-EM decrease at lower voltage, leading to loss of SNR. Here, we present an electron counting algorithm to improve the detection of low-energy electrons. The counting algorithm increased the SNR of 120-kV and 200-kV cryo-EM image from a Falcon III camera by 8%, 20% at half the Nyquist frequency and 21%, 80% at Nyquist frequency, respectively, resulting in a considerable improvement in resolution of 3D reconstructions. Our results indicate that with further improved temporal coherence and a dedicated designed camera, a 120-kV cryo-electron microscope has potential to match the 300-kV microscope at imaging small proteins.

Low-cooling-rate freezing in biomolecular cryo-electron microscopy for recovery of initial frames.

When biological samples are first exposed to electrons in cryo-electron microcopy (cryo-EM), proteins exhibit a rapid 'burst' phase of beam-induced motion that cannot be corrected with software. This lowers the quality of the initial frames, which are the least damaged by the electrons. Hence, they are commonly excluded or down-weighted during data processing, reducing the undamaged signal and the resolution in the reconstruction. By decreasing the cooling rate during sample preparation, either with a cooling-rate gradient or by increasing the freezing temperature, we show that the quality of the initial frames for various protein and virus samples can be recovered. Incorporation of the initial frames in the reconstruction increases the resolution by an amount equivalent to using ~60% more data. Moreover, these frames preserve the high-quality cryo-EM densities of radiation-sensitive residues, which is often damaged or very weak in canonical three-dimensional reconstruction. The improved freezing conditions can be easily achieved using existing devices and enhance the overall quality of cryo-EM structures.

Structural Insight into Phospholipid Transport by the MlaFEBD Complex from P. aeruginosa.

The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4-3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaF2E2B2D6, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.

Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump.

The oxaloacetate decarboxylase sodium pump (OAD) is a unique primary-active transporter that utilizes the free energy derived from oxaloacetate decarboxylation for sodium transport across the cell membrane. It is composed of 3 subunits: the α subunit catalyzes carboxyl-transfer from oxaloacetate to biotin, the membrane integrated ß subunit catalyzes the subsequent carboxyl-biotin decarboxylation and the coupled sodium transport, the γ subunit interacts with the α and ß subunits and stabilizes the OAD complex. We present here structure of the Salmonella typhimurium OAD ßγ sub-complex. The structure revealed that the ß and γ subunits form a ß3γ3 hetero-hexamer with extensive interactions between the subunits and shed light on the OAD holo-enzyme assembly. Structure-guided functional studies provided insights into the sodium binding sites in the ß subunit and the coupling between carboxyl-biotin decarboxylation and sodium transport by the OAD ß subunit.

Conformational activation of ribosome recycling by intra- and inter-molecular dynamics of RRF.

Ribosome recycling is the final step of the cyclic process of translation, where the post-termination complex (PoTC) is disassembled by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in the sub-second time range. Since, however, both the RRF and PoTC display highly dynamic action during this process, it is difficult to assess the molecular details of the interactions between the factors and the ribosome that are essential for rapid subunit separation. Here we characterized the molecular dynamics of RRF and PoTC by combined use of molecular dynamics simulations, single molecule fluorescence detection and single-particle cryo-EM analysis, with time resolutions in the sub-millisecond to minute range. We found that RRF displays two-layer dynamics: intra- and inter-molecular dynamics during ribosome splitting. The intra-molecular dynamics exhibits two different configurations of RRF: 'bent' and 'extended'. A single-site mutant of RRF increases its propensity to the 'extended' conformation and leads to a higher binding affinity of RRF to the PoTC. The inter-molecular dynamics between RRF and EF-G in the PoTC reveals that the domain IV of EF-G pushes against the domain II of RRF, triggering the disruption of the major inter-subunit bridge B2a, and catalyzes the splitting.

Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG.

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB2FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB2FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic ß-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the ß-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

Vacancy-mediated magnetism in pure copper oxide nanoparticles.

Room temperature ferromagnetism (RTF) is observed in pure copper oxide (CuO) nanoparticles which were prepared by precipitation method with the post-annealing in air without any ferromagnetic dopant. X-ray photoelectron spectroscopy (XPS) result indicates that the mixture valence states of Cu1+ and Cu2+ ions exist at the surface of the particles. Vacuum annealing enhances the ferromagnetism (FM) of CuO nanoparticles, while oxygen atmosphere annealing reduces it. The origin of FM is suggested to the oxygen vacancies at the surface/or interface of the particles. Such a ferromagnet without the presence of any transition metal could be a very good option for a class of spintronics.