Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time- and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy (cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence (AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of medium-resolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.

Pannexin 2 (Panx2) is a large-pore ATP-permeable channel with critical roles in various physiological processes, such as the inflammatory response, energy production and apoptosis. Its dysfunction is related to numerous pathological conditions including ischemic brain injury, glioma and glioblastoma multiforme. However, the working mechanism of Panx2 remains unclear. Here, we present the cryo-electron microscopy structure of human Panx2 at a resolution of 3.4 Å. Panx2 structure assembles as a heptamer, forming an exceptionally wide channel pore across the transmembrane and intracellular domains, which is compatible with ATP permeation. Comparing Panx2 with Panx1 structures in different states reveals that the Panx2 structure corresponds to an open channel state. A ring of seven arginine residues located at the extracellular entrance forms the narrowest site of the channel, which serves as the critical molecular filter controlling the permeation of substrate molecules. This is further verified by molecular dynamics simulations and ATP release assays. Our studies reveal the architecture of the Panx2 channel and provide insights into the molecular mechanism of its channel gating.

The bacterial flagellar motor is a self-assembling supramolecular nanodevice. Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the N-terminal domain of FliG (FliF_C-FliG_N) from the bacterium Helicobacter pylori. Within this complex, FliF_C interacted with FliG_N through extensive hydrophobic contacts similar to those observed in the FliF_C-FliG_N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF_C-FliG_N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo-repeat-like motifs (ARM) of FliG_N. Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG_N stacking by complementation studies in Escherichia coli. Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG_N, containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.

Human gastric pathogen Helicobacter pylori ( H. pylori ) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed β-helix made up of two domains with extensive domain–domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.