Cryo-EM reveals the mechanism of DNA compaction by Mycobacterium smegmatis Dps2.

DNA binding protein from starved cells (Dps) is a miniature ferritin complex, which plays a vital role in protecting bacterial DNA during starvation to maintain the integrity of bacteria under hostile conditions. Several approaches, including cryo-electron tomography, have been previously implemented by other research groups to decipher the structure of the Dps protein bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to identify the DNA binding residues. Like other bacteria, Mycobacterium smegmatis alsoexpresses Dps2 (called MsDps2), which binds DNA to protect it under oxidative stress conditions. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps2 protein from Mycobacterium smegmatis. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA complexes and identified that the region close to the N-terminus confers the DNA binding property. Based on cryo-EM data, we also pinpointed several arginine residues, proximal to the DNA binding region, responsible for DNA binding. We also performed mutations of these residues, which dramatically reduced the MsDps2-DNA interaction. In addition, we proposed a model that elucidates the mechanism of DNA compaction, which adapts a lattice-like structure. We performed single-molecule imaging of MsDps2-DNA interactions that corroborate well with our structural studies. Taken together, our results delineate the specific MsDps2 residues that play an important role in DNA binding and compaction, providing new insights into Mycobacterial DNA compaction mechanisms under stress conditions.

Multiscale Materials Engineering via Self-Assembly of Pentapeptide Derivatives from SARS CoV E Protein.

Short peptide-based supramolecular hydrogels hold enormous potential for a wide range of applications. However, the gelation of these systems is very challenging to control. Minor changes in the peptide sequence can significantly influence the self-assembly mechanism and thereby the gelation propensity. The involvement of SARS CoV E protein in the assembly and release of the virus suggests that it may have inherent self-assembling properties that can contribute to the development of hydrogels. Here, three pentapeptide sequences derived from C-terminal of SARS CoV E protein are explored with same amino acid residues but different sequence distributions and discovered a drastic difference in the gelation propensity. By combining spectroscopic and microscopic techniques, the relationship between peptide sequence arrangement and molecular assembly structure are demonstrated, and how these influence the mechanical properties of the hydrogel. The present study expands the variety of secondary structures for generating supramolecular hydrogels by introducing the 310-helix as the primary building block for gelation, facilitated by a water-mediated structural transition into ß-sheet conformation. Moreover, these Fmoc-modified pentapeptide hydrogels/supramolecular assemblies with tunable morphology and mechanical properties are suitable for tissue engineering, injectable delivery, and 3D bio-printing applications.

Practical synthesis of isoindolines yields potent colistin potentiators for multidrug-resistant Acinetobacter baumannii.

An efficient and practical one-pot synthesis of isoindolines from readily available starting materials was achieved under mild conditions by implementing an isoindole umpolung strategy. A variety of isoindolines were prepared with good to excellent yields. Biological screens of these identified compounds demonstrated that they are potent potentiators of colistin for multi-drug resistant Acinetobacter baumannii.

Cryo-EM research in India.

To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.

Erratum: Recognition determinants of improved HIV-1 neutralization by a heavy chain matured pediatric antibody.

[This corrects the article DOI: 10.1016/j.isci.2023.107579.].

Recognition determinants of improved HIV-1 neutralization by a heavy chain matured pediatric antibody.

The structural and characteristic features of HIV-1 broadly neutralizing antibodies (bnAbs) from chronically infected pediatric donors are currently unknown. Herein, we characterized a heavy chain matured HIV-1 bnAb 44m, identified from a pediatric elite-neutralizer. Interestingly, in comparison to its wild-type AIIMS-P01 bnAb, 44m exhibited moderately higher level of somatic hypermutations of 15.2%. The 44m neutralized 79% of HIV-1 heterologous viruses (n = 58) tested, with a geometric mean IC50 titer of 0.36 µg/mL. The cryo-EM structure of 44m Fab in complex with fully cleaved glycosylated native-like BG505.SOSIP.664.T332N gp140 envelope trimer at 4.4 Å resolution revealed that 44m targets the V3-glycan N332-supersite and GDIR motif to neutralize HIV-1 with improved potency and breadth, plausibly attributed by a matured heavy chain as compared to that of wild-type AIIMS-P01. This study further improves our understanding on pediatric HIV-1 bnAbs and structural basis of broad HIV-1 neutralization by 44m may be useful blueprint for vaccine design in future.

Cryo-EM-based structural insights into supramolecular assemblies of γ-hemolysin from S. aureus reveal the pore formation mechanism.

γ-Hemolysin (γ-HL) is a hemolytic and leukotoxic bicomponent ß-pore-forming toxin (ß-PFT), a potent virulence factor from the Staphylococcus aureus Newman strain. In this study, we performed single-particle cryoelectron microscopy (cryo-EM) of γ-HL in a lipid environment. We observed clustering and square lattice packing of octameric HlgAB pores on the membrane bilayer and an octahedral superassembly of octameric pore complexes that we resolved at resolution of 3.5 Å. Our atomic model further demonstrated the key residues involved in hydrophobic zipping between the rim domains of adjacent octameric complexes, providing additional structural stability in PFTs post oligomerization. We also observed extra densities at the octahedral and octameric interfaces, providing insights into the plausible lipid-binding residues involved for HlgA and HlgB components. Furthermore, the hitherto elusive N-terminal region of HlgA was also resolved in our cryo-EM map, and an overall mechanism of pore formation for bicomponent ß-PFTs is proposed.

Cryo-EM reveals the membrane-binding phenomenon of EspB, a virulence factor of the mycobacterial type VII secretion system.

Mycobacterium tuberculosis (Mtb) utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. EspB, a ∼36 kDa secreted substrate of the ESX-1 apparatus, was shown to cause ESAT-6-independent host cell death. Despite the current wealth of high-resolution structural information of the ordered N-terminal domain, the mechanism of EspB-mediated virulence remains poorly characterized. Here, we document EspB interaction with phosphatidic acid (PA) and phosphatidylserine (PS) in the context of membranes, through a biophysical approach including transmission electron microscopy and cryo-EM. We were also able to show PA, PS-dependent conversion of monomers to oligomers at physiological pH. Our data suggest that EspB adheres to biological membranes with limited PA and PS. EM of yeast mitochondria with EspB indicates a mitochondrial membrane-binding property of this ESX-1 substrate. Further, we determined the 3D structures of EspB with and without PA and observed plausible stabilization of the low complexity C-terminal domain in the presence of PA. Collectively, our cryo-EM-based structural and functional studies of EspB provide further insight into the host-Mtb interaction.

Neutralizing Efficacy of Encapsulin Nanoparticles against SARS-CoV2 Variants of Concern.

Rapid emergence of the SARS-CoV-2 variants has dampened the protective efficacy of existing authorized vaccines. Nanoparticle platforms offer a means to improve vaccine immunogenicity by presenting multiple copies of desired antigens in a repetitive manner which closely mimics natural infection. We have applied nanoparticle display combined with the SpyTag-SpyCatcher system to design encapsulin-mRBD, a nanoparticle vaccine displaying 180 copies of the monomeric SARS-CoV-2 spike receptor-binding domain (RBD). Here we show that encapsulin-mRBD is strongly antigenic and thermotolerant for long durations. After two immunizations, squalene-in-water emulsion (SWE)-adjuvanted encapsulin-mRBD in mice induces potent and comparable neutralizing antibody titers of 105 against wild-type (B.1), alpha, beta, and delta variants of concern. Sera also neutralizes the recent Omicron with appreciable neutralization titers, and significant neutralization is observed even after a single immunization.

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM.

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.