Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase.

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Enhanced treatment of synthetic wastewater by bioaugmentation with a constructed consortium.

Bioaugmentation by adding well-functioning mixed microorganism consortia represents a potentially useful approach to improve contaminant removal in wastewater treatment plants (WWTPs). However, unfavorable environmental conditions (i.e., low temperatures) can severely inhibit microbial activity, drawing our attention to constructing cold-tolerant microorganism preparations and investigating their availability in practical applications. Here we screened four in situ functional isolates from the activated sludge of secondary sedimentation tanks in WWTPs to construct a psychrophilic microbial consortium, which was used to perform bioaugmentation for enhanced removal of nitrogen and phosphorus under low temperatures. The consortium was established by cocultivation of four isolates, characterized by 16 S rRNA as the COD-degrading bacterium Aeromonas sp. Z3, aerobic denitrifying bacterium Acinetobacter sp. HF9, nitrifying bacterium Klebsiella sp. X8, and polyphosphate-accumulating bacterium Pseudomonas sp. PC5 respectively. The microorganism preparation was composed of Z3, HF9, X8, and PC5 under the ratio of 1: 1: 3: 1, which can exert optimal pollutant removal under the conditions of 12 °C, 6.0-9.0 pH, 120-200 r‧min-1, and a dosage of 5% (V/V). A 30-day continuous operation of the bioaugmented and control sequencing batch reactors (SBRs) was investigated, and the bioaugmented SBR showed a shorter start-up stage and a more stable operating situation. Compared to the control SBR, the COD, NH4+-N, TN, and TP removal efficiency of the bioaugmented SBR increased by an average of 7.95%, 9.05%, 9.54%, and 7.45% respectively. The analysis of the microbial community revealed that the introduced isolates were dominant in the activated sludge and that functional taxa such as Proteobacteria, Bacteroidota, and Actinobacteria were further enriched after a period of bioaugmentation. The study provides some basis and guidance for the practical application of how to strengthen the stable operation of WWTPs under low temperatures.

Enhanced visible light photo-Fenton catalysis by lanthanum-doping BiFeO3 for norfloxacin degradation.

Efficient photo-Fenton removal of antibiotic effluent is a widely followed and significant attempt to deal with the growing environmental pollution. In this study, BiFeO3 and lanthanum doped BiFeO3 catalysts were synthesized via one-step hydrothermal method as hydrogen peroxide activator for mineralization of norfloxacin (NOR). Various characterization measurements were used to verify La was successfully doped into the lattice of perovskite and investigated the effect of La doping molar ratio on BiFeO3 through the characterization of the morphology and physicochemical properties. The degradation experiment and reaction rate constants showed that the La-doped BiFeO3 particle exhibited superior photo-Fenton catalytic performance to undoped BiFeO3. Especially, the degradation efficiency of 15% La-doped BiFeO3 could reach up to 84.94%. And the first order kinetic constant of optimized conditions was 0.01638 min-1, which was about 6.9 times than that of undoped BiFeO3.The influence of pH, oxidizer content and catalyst dosage in photo-Fenton reaction were investigated detailedly. Besides, the synthetic catalyst possessed favorable stability and reusability with little metal leaching after many cycles of use. Radical scavenger experiments and electron spin resonance tests were carried out to conclude that the ·OH and holes were regarded as the dominate active species in the catalytic process. The narrow band gap and excellent electron transfer efficiency were the key factors for La-doped BiFeO3 to have high catalytic efficiency in the photo-Fenton system. Current works demonstrated the great promise of La-doped BiFeO3 in the elimination of antibiotic organics.

Multivalent interactions essential for lentiviral integrase function.

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity.

Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca2+ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.

Crystal structure of the coiled-coil domain of Drosophila TRIM protein Brat.

Drosophila brain tumor (Brat) is a translational repressor belonging to the tripartite motif (TRIM) protein superfamily. During the asymmetric division of Drosophila neuroblasts, Brat localizes at the basal cortex via direct interaction with the scaffolding protein Miranda (Mira), and segregates into the basal ganglion mother cells after cell division. It was previously reported that both the coiled-coil (CC) and NHL domains of Brat are required for the interaction with Mira, but the underlying structural basis is elusive. Here, we determine the crystal structure of Brat-CC domain (aa 376-511) at 2.5 Å, showing that Brat-CC forms an elongated antiparallel dimer through an unconventional CC structure. The dimeric assembly in Brat-CC structure is similar to its counterparts in other TRIM proteins, but Brat-CC also exhibits some distinct structural features. We also demonstrate that the CC domain could not bind Mira by its own, neither does the isolated NHL domain of Brat. Rather, Brat binds to Mira through the CC-NHL domain tandem, indicating that the function of the CC domain is to assemble Brat-NHL in dimeric form, which is necessary for Mira binding.

WW domain-mediated regulation and activation of E3 ubiquitin ligase Suppressor of Deltex.

The Nedd4 family E3 ligases Itch and WWP1/2 play crucial roles in the regulation of cell cycle progression and apoptosis and are closely correlated with cancer development and metastasis. It has been recently shown that the ligase activities of Itch and WWP1/2 are tightly regulated, with the HECT domain sequestered intramolecularly by a linker region connecting WW2 and WW3. Here, we show that a similar autoinhibitory mechanism is utilized by the Drosophila ortholog of Itch and WWP1/2, Suppressor of Deltex (Su(dx)). We show that Su(dx) adopts an inactive steady state with the WW domain region interacting with the HECT domain. We demonstrate that both the linker and preceding WW2 are required for the efficient binding and regulation of Su(dx) HECT. Recruiting the multiple-PY motif-containing adaptor dNdfip via WW domains relieves the inhibitory state of Su(dx) and leads to substrate (e.g. Notch) ubiquitination. Our study demonstrates an evolutionarily conservative mechanism governing the regulation and activation of some Nedd4 family E3 ligases. Our results also suggest a dual regulatory mechanism for specific Notch down-regulation via dNdfip-Su(dx)-mediated Notch ubiquitination.

Basal condensation of Numb and Pon complex via phase transition during Drosophila neuroblast asymmetric division.

Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) asymmetric cell divisions and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentiation. Here we discover that Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. The multivalent Numb-Pon interaction leads to high binding specificity and liquid-liquid phase separation of the complex. Perturbations of the Numb/Pon complex phase transition impair the basal localization of Numb and its subsequent suppression of Notch signaling during NB asymmetric divisions. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes.

Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·α-adaptin complex.

Traffic of cargo across membranes helps establish, maintain, and reorganize distinct cellular compartments and is fundamental to many metabolic processes. The cargo-selective endocytic adaptor Numb participates in clathrin-dependent endocytosis by attaching cargoes to the clathrin adaptor α-adaptin. The phosphorylation of Numb at Ser265 and Ser284 recruits the regulatory protein 14-3-3, accompanied by the dissociation of Numb from α-adaptin and Numb's translocation from the cortical membrane to the cytosol. However, the molecular mechanisms underlying the Numb-α-adaptin interaction and its regulation by Numb phosphorylation and 14-3-3 recruitment remain poorly understood. Here, biochemical and structural analyses of the Numb·14-3-3 complex revealed that Numb phosphorylation at both Ser265 and Ser284 is required for Numb's efficient interaction with 14-3-3. We also discovered that an RQFRF motif surrounding Ser265 in Numb functions together with the canonical C-terminal DPF motif, required for Numb's interaction with α-adaptin, to form a stable complex with α-adaptin. Of note, we provide evidence that the phosphorylation-induced binding of 14-3-3 to Numb directly competes with the binding of α-adaptin to Numb. Our findings suggest a potential mechanism governing the dynamic assembly of Numb with α-adaptin or 14-3-3. This dual-site recognition of Numb by α-adaptin may have implications for other α-adaptin targets. We propose that the newly identified α-adaptin-binding site surrounding Ser265 in Numb functions as a triggering mechanism for the dynamic dissociation of the Numb·α-adaptin complex.

Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch.

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto-inhibition. However, the molecular mechanism underlying Nedd4 E3 auto-inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2-E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1-mediated phosphorylation relieves the auto-inhibition of Itch in a WW2-dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.

Phospho-Pon Binding-Mediated Fine-Tuning of Plk1 Activity.

In Drosophila neuroblasts (NBs), the asymmetrical localization and segregation of the cell-fate determinant Numb are regulated by its adaptor Partner of Numb (Pon) and the cell-cycle kinase Polo. Polo phosphorylates the Pon localization domain, thus leading to its basal distribution together with Numb, albeit through an unclear mechanism. Here, we find that Cdk1 phosphorylates Pon at Thr63, thus creating a docking site for the Polo-box domain (PBD) of Polo-like kinase 1 (Plk1). The crystal structure of the Plk1 PBD/phospho-Pon complex reveals that two phospho-Pon bound PBDs associate to form a dimer of dimers. We provide evidence that phospho-Pon binding-induced PBD dimerization relieves the autoinhibition of Plk1. Moreover, we demonstrate that the priming Cdk1 phosphorylation of Pon is important for sequential Plk1 phosphorylation. Our results not only provide structural insight into how phosphoprotein binding activates Plk1 but also suggest that binding to different phosphoproteins might mediate the fine-tuning of Plk1 activity.

The structural basis of Miranda-mediated Staufen localization during Drosophila neuroblast asymmetric division.

During the asymmetric division of Drosophila neuroblasts (NBs), the scaffold Miranda (Mira) coordinates the subcellular distribution of cell-fate determinants including Staufen (Stau) and segregates them into the ganglion mother cells (GMCs). Here we show the fifth double-stranded RNA (dsRNA)-binding domain (dsRBD5) of Stau is necessary and sufficient for binding to a coiled-coil region of Mira cargo-binding domain (CBD). The crystal structure of Mira514-595/Stau dsRBD5 complex illustrates that Mira forms an elongated parallel coiled-coil dimer, and two dsRBD5 symmetrically bind to the Mira dimer through their exposed ß-sheet faces, revealing a previously unrecognized protein interaction mode for dsRBDs. We further demonstrate that the Mira-Stau dsRBD5 interaction is responsible for the asymmetric localization of Stau during Drosophila NB asymmetric divisions. Finally, we find the CBD-mediated dimer assembly is likely a common requirement for Mira to recognize and translocate other cargos including brain tumour (Brat).