Ning Gao

Controlling Assembly of Paired Gold Clusters within Apoferritin Nanoreactor for in Vivo Kidney Targeting and Biomedical Imaging

Functional nanostructures with high biocompatibility and stability, low toxicity, and specificity of targeting to desired organs or cells are of great interest in nanobiology and medicine. However, the challenge is to integrate all of these desired features into a single nanobiostructure, which can be applied to biomedical applications and eventually in clinical settings. In this context, we designed a strategy to assemble two gold nanoclusters at the ferroxidase active sites of ferritin heavy chain. Our studies showed that the resulting nanostructures (Au-Ft) retain not only the intrinsic fluorescence properties of noble metal, but gain enhanced intensity, show a red-shift, and exhibit tunable emissions due to the coupling interaction between the paired Au clusters. Furthermore, Au-Ft possessed the well-defined nanostructure of native ferritin, showed organ-specific targeting ability, high biocompatibility, and low cytotoxicity. The current study demonstrates that an integrated multimodal assembly strategy is able to generate stable and effective biomolecule-noble metal complexes of controllable size and with desirable fluorescence emission characteristics. Such agents are ideal for targeted in vitro and in vivo imaging. These results thus open new opportunities for biomolecule-guided nanostructure assembly with great potential for biomedical applications.

Architecture of the mammalian mechanosensitive Piezo1 channel

Piezo proteins are evolutionarily conserved and functionally diverse mechanosensitive cation channels. However, the overall structural architecture and gating mechanisms of Piezo channels have remained unknown. Here we determine the cryo-electron microscopy structure of the full-length (2,547 amino acids) mouse Piezo1 (Piezo1) at a resolution of 4.8 Å. Piezo1 forms a trimeric propeller-like structure (about 900 kilodalton), with the extracellular domains resembling three distal blades and a central cap. The transmembrane region has 14 apparently resolved segments per subunit. These segments form three peripheral wings and a central pore module that encloses a potential ion-conducting pore. The rather flexible extracellular blade domains are connected to the central intracellular domain by three long beam-like structures. This trimeric architecture suggests that Piezo1 may use its peripheral regions as force sensors to gate the central ion-conducting pore.

Structure of the eukaryotic MCM complex at 3.8 Å

DNA replication in eukaryotes is strictly regulated by several mechanisms. A central step in this replication is the assembly of the heterohexameric minichromosome maintenance (MCM2–7) helicase complex at replication origins during G1 phase as an inactive double hexamer. Here, using cryo-electron microscopy, we report a near-atomic structure of the MCM2–7 double hexamer purified from yeast G1 chromatin. Our structure shows that two single hexamers, arranged in a tilted and twisted fashion through interdigitated amino-terminal domain interactions, form a kinked central channel. Four constricted rings consisting of conserved interior β-hairpins from the two single hexamers create a narrow passageway that tightly fits duplex DNA. This narrow passageway, reinforced by the offset of the two single hexamers at the double hexamer interface, is flanked by two pairs of gate-forming subunits, MCM2 and MCM5. These unusual features of the twisted and tilted single hexamers suggest a concerted mechanism for the melting of origin DNA that requires structural deformation of the intervening DNA.

The crystal structure of Cpf1 in complex with CRISPR RNA.

The CRISPR–Cas systems, as exemplified by CRISPR–Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection. The CRISPR–Cpf1 system, a new class 2 CRISPR–Cas system, mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38u2009Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)2+ ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.

Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein.

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6xa0Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

The architecture of the mammalian respirasome

The respiratory chain complexes I, III and IV (CI, CIII and CIV) are present in the bacterial membrane or the inner mitochondrial membrane and have a role of transferring electrons and establishing the proton gradient for ATP synthesis by complex V. The respiratory chain complexes can assemble into supercomplexes (SCs), but their precise arrangement is unknown. Here we report a 5.4u2009Å cryo-electron microscopy structure of the major 1.7 megadalton SCI1III2IV1 respirasome purified from porcine heart. The CIII dimer and CIV bind at the same side of the L-shaped CI, with their transmembrane domains essentially aligned to form a transmembrane disk. Compared to free CI, the CI in the respirasome is more compact because of interactions with CIII and CIV. The NDUFA11 and NDUFB9 supernumerary subunits of CI contribute to the oligomerization of CI and CIII. The structure of the respirasome provides information on the precise arrangements of the respiratory chain complexes in mitochondria.

Structure of a Pancreatic ATP-Sensitive Potassium Channel

ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closedxa0conformation. In another structural population, axa0putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.

CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane

A fundamental question regarding autophagosome formation is how the shape of the double-membrane autophagosomal vesicle is generated. Here we show that in mammalian cells assembly of an actin scaffold inside the isolation membrane (the autophagosomal precursor) is essential for autophagosomal membrane shaping. Actin filaments are depolymerized shortly after starvation and actin is assembled into a network within the isolation membrane. When formation of actin puncta is disrupted by an actin polymerization inhibitor or by knocking down the actin-capping protein CapZβ, isolation membranes and omegasomes collapse into mixed-membrane bundles. Formation of actin puncta is PtdIns(3)P dependent, and inhibition of PtdIns(3)P formation by treating cells with the PI(3)K inhibitor 3-MA, or by knocking down Beclin-1, abolishes the formation of actin puncta. Binding of CapZ to PtdIns(3)P, which is enriched in omegasomes, stimulates actin polymerization. Our findings illuminate the mechanism underlying autophagosomal membrane shaping and provide key insights into how autophagosomes are formed.

Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase

Kinetics and cryo-electronmicroscopy data provide insights into GTPase ObgE’s role as a ribosome anti-association factor that is modulated by nutrient availability, coupling growth control to ribosome biosynthesis and protein translation.

Structural basis for the function of a small GTPase RsgA on the 30S ribosomal subunit maturation revealed by cryoelectron microscopy

The bacterial RsgA, a circularly permutated GTPase, whose GTPase activity is dependent on the 30S ribosomal subunit, is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation. The role of RsgA is to release another 30S biogenesis factor, RbfA, from the mature 30S subunit in a GTP-dependent manner. Using cryoelectron microscopy, we have determined the structure of the 30S subunit bound with RsgA in the presence of GMPPNP at subnanometer resolution. In the structure, RsgA binds to the central part of the 30S subunit, close to the decoding center, in a position that is incompatible with multiple biogenesis factors, all three translation initiation factors, as well as A-, P-site tRNAs and the 50S subunit. Further structural analysis not only provides a structural model for the RsgA-dependent release of RbfA from the nascent 30S subunit, but also indicates RsgA’s role in the ribosomal protein assembly, to promote some tertiary binding protein incorporation. Moreover, together with available biochemical and genetic data, our results suggest that RsgA might be a general checkpoint protein in the late stage of the 30S subunit biogenesis, whose function is not only to release biogenesis factors (e.g., RbfA) from the nascent 30S subunit, but also to block the association of initiation factors to the premature 30S subunit.