Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fasciculation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains. Here, we show that CNTN2 forms transient homophilic interactions (KD ∼200 nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers. Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side of the homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometry and cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediating homodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mechanisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio of protein interactions and that can be leveraged to strategically guide neural circuit development.

Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Å or better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42 Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48 h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for ∼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5 Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump\'s structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump\'s assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.

Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.

Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.

ABSTRACTSARS-CoV-2 has been evolving into a large number of variants, including the highly pathogenic Delta variant, and the currently prevalent Omicron subvariants with extensive evasion capability, which raises an urgent need to develop new broad-spectrum neutralizing antibodies. Herein, we engineer two IgG-(scFv)2 form bispecific antibodies with overlapping epitopes (bsAb1) or non-overlapping epitopes (bsAb2). Both bsAbs are significantly superior to the parental monoclonal antibodies in terms of their antigen-binding and virus-neutralizing activities against all tested circulating SARS-CoV-2 variants including currently dominant JN.1. The bsAb1 can efficiently neutralize all variants insensitive to parental monoclonal antibodies or the cocktail with IC50 lower than 20 ng/mL, even slightly better than bsAb2. Furthermore, the cryo-EM structures of bsAb1 in complex with the Omicron spike protein revealed that bsAb1 with overlapping epitopes effectively locked the S protein, which accounts for its conserved neutralization against Omicron variants. The bispecific antibody strategy engineered from overlapping epitopes provides a novel solution for dealing with viral immune evasion.

Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor (PAFR). However, drug development targeting the PAFR has been limited, partly due to an incomplete understanding of its activation mechanism. Here, we present a 2.9-Å structure of the PAF-bound PAFR-Gi complex. Structural and mutagenesis analyses unveil a specific binding mode of PAF, with the choline head forming cation-π interactions within PAFR hydrophobic pocket, while the alkyl tail penetrates deeply into an aromatic cleft between TM4 and TM5. Binding of PAF modulates conformational changes in key motifs of PAFR, triggering the outward movement of TM6, TM7, and helix 8 for G protein coupling. Molecular dynamics simulation suggests a membrane-side pathway for PAF entry into PAFR via the TM4-TM5 cavity. By providing molecular insights into PAFR signaling, this work contributes a foundation for developing therapeutic interventions targeting PAF signal axis.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.