The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB₃, EccC₃, and EccE₃ and two copies of the EccD₃ protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.

Human serum albumin (HSA) is the most prevalent plasma protein in the human body, accounting for 60 % of the total plasma protein. HSA plays a major pharmacokinetic function, serving as a facilitator in the distribution of endobiotics and xenobiotics within the organism. In this paper we report the cryoEM structures of HSA in the apo form and in complex with two ligands (salicylic acid and teniposide) at a resolution of 3.5, 3.7 and 3.4 Å, respectively. We expand upon previously published work and further demonstrate that sub-4 Å maps of ∼60 kDa proteins can be routinely obtained using a 200 kV microscope, employing standard workflows. Most importantly, these maps allowed for the identification of small molecule ligands, emphasizing the practical applicability of this methodology and providing a starting point for subsequent computational modeling and in silico optimization.

The Sec61 translocon mediates entry of most secreted and transmembrane proteins into the endoplasmic reticulum, providing a novel therapeutic target to block the expression of protumorigenic factors. Sec61 inhibitors with antitumor activity, mostly derived from natural products, have been reported. However, poor tolerability and suboptimal pharmaceutical properties have precluded their further development. We report here the discovery and characterization of KZR-834 and KZR-261, related small molecule analogs that directly bind to the Sec61 channel to potently inhibit the biogenesis of a subset of Sec61 client proteins. This client inhibition profile includes several tumorigenic factors, results in the activation of an endoplasmic reticulum stress response, and leads to broad anticancer effects in vitro. In vivo, KZR-261 was well tolerated and exhibits antitumor effects across multiple models, both as a single agent and in combination with anti-PD-1 immunotherapy. Based on the strength of this preclinical data, KZR-261 progressed into a phase I clinical trial (NCT05047536) in patients with malignant disease, where it was found to be well tolerated at doses that achieved durable stable disease. These results highlight the potential of Sec61 inhibition as a novel therapeutic target. SIGNIFICANCE STATEMENT: KZR-834 and KZR-261 are novel Sec61 inhibitors with the ability to block multiple Sec61 client proteins, leading to well-tolerated efficacy in in vivo cancer models. This represents a novel mechanism for blocking expression of oncogenic factors, including those not amenable to targeting through conventional methods.

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins’ native conformations, thus affecting stability and functionality. This study leverages single-particle cryo-electron microscopy to elucidate the structural nuances of membrane proteins, focusing on the SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) purified with diverse detergents, including n-dodecyl β-D-maltopyranoside (DDM), glycodiosgenin (GDN), β-D-octyl-glucoside (OG), and lauryl maltose neopentyl glycol (LMNG). This research not only contributes to the understanding of membrane protein structures but also addresses detergent effects on protein purification. By showcasing that the overall structural integrity of the channel is preserved, our study underscores the intricate interplay between proteins and detergents, offering insightful implications for drug design and membrane biology.

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane; however, the architecture and mechanism of translocation has remained elusive. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the model organism Mycobacterium smegmatis . The structure reveals two large α -helical membrane pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric cytoplasmic domain is poised to gate and regulate the pore’s function. Our study provides mechanistic insights into the ESX systems and will guide structure-based design of drugs targeting this unique bacterial translocon. One Sentence Summary: The structure of the ESX-3 secretion system reveals a pore of sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory apparatus.

Different manufacturing approaches have been used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Sf9 insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed for the first time a highly controlled comparative production analysis varying only the host cell species but keeping all other rAAV production parameters the same. We demonstrate that host cell species is critical for determining vector potency. Given these key findings, we then sought to deeply characterize differences in rAAVs when produced by these two manufacturing platforms with multiple analytical approaches including: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and comparative functional transduction assessments in vitro and in vivo , including in humanized liver mice. Using these tools we’ve made two major discoveries: 1) rAAV capsids have post-translational modifications (PTMs) including glycosylation, acetylation, phosphorylation, methylation and deamidation, and these PTMs differ between platforms; 2) rAAV genomes are methylated during production, and these methylation marks are also differentially deposited between platforms. In addition, our data also demonstrate that host cell protein impurities differ between platforms and can have their own PTMs including potentially immunogenic N-linked glycans. We show that human-produced rAAVs are more potent than baculovirus- Sf9 vectors in various cell types in vitro ( P < 0.05-0.0001), in various mouse tissues in vivo ( P < 0.03-0.0001), and in human liver in vivo ( P < 0.005). Collectively, our findings were reproducible across vendors, including commercial manufacturers, academic core facilities, and individual laboratory preparations. These vector differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity as well as cost considerations.