Summary Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via Type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically, but rather by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps explain interdependence of substrates and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.

ngaging the mostly undruggable proteome to uncover new disease therapies not only requires technological innovations that facilitate rapid discovery of ligandable hotspots across the proteome but also demands new therapeutic modalities that alter protein function through novel mechanisms 1,2 . Targeted protein degradation (TPD) tackles the undruggable proteome by targeting specific proteins for ubiquitination and proteasomal degradation. One major class of small-molecule effectors of TPD, proteolysis-targeting chimeras (PROTACs), are heterobifunctional molecules that consist of an E3 ligase recruiter linked to a protein-targeting ligand to induce the formation of ternary complexes that bring together an E3 ubiquitin ligase and the target protein as a neo-substrate [3][4][5] . PROTACs have enabled the targeted and specific degradation of numerous disease-causing proteins in cells 3,6 . New approaches for TPD have also arisen that exploit endosomal and lysosomal degradation pathways with lysosome-targeting chimeras or autophagy with autophagy-targeting chimeras 7,8 . New approaches for chemically induced proximity beyond degradation have also been developed in recent years, including targeted phosphorylation with phosphorylation-inducing chimeric small molecules and targeted dephosphorylation, but no small-molecule-based induced proximity approaches exist for targeted deubiquitination and subsequent stabilization of proteins 9,10 .Active ubiquitination and degradation of proteins is the root cause of several classes of diseases, including many tumor suppressors in cancer (for example, TP53, CDKN1A, CDN1C and BAX), and mutated and misfolded proteins, such as ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis or glucokinase in pancreatic cells in maturity-onset diabetes of the young type 2. In these cases, a TPS therapeutic strategy, rather than degradation, would be beneficial [11][12][13][14] . Analogous to TPD, we hypothesized that TPS could be enabled by the discovery of a small-molecule recruiter of a deubiquitinase (DUB) that could be linked to a protein-targeting ligand to form a chimeric molecule, which would induce the deubiquitination and stabilization of proteins of interest. We call this heterobifunctional stabilizer a DUBTAC (Fig. 1a). In this study, we report the discovery of a covalent recruiter for the K48-ubiquitin chain-specific DUB OTUB1, which when linked to a protein-targeting ligand stabilizes an actively degraded target protein to demonstrate proof of concept for the DUBTAC platform. ResultsIdentifying allosteric ligandable sites within DUBs. To enable the DUBTAC platform, our first goal was to identify a small-molecule recruiter that targeted an allosteric site on a DUB without inhibiting DUB function, as the recruitment of a functional DUB would be required to deubiquitinate and stabilize the target protein. While many DUBs possess well-defined active sites bearing a catalytic and highly nucleophilic cysteine, there have not yet been systematic evaluations of ...

Background: Septins self-assemble into hetero-octameric rods and higher order structures and recruit other proteins.Results: A spectroscopic method (FRET) to measure septin interactions and binding of associated proteins was developed.Conclusion: End to end polymerization of Cdc11-capped rods, heterotypic end to end junctions between Cdc11-capped rods and Shs1-capped rods, and binding of an associated protein were demonstrated.Significance: This spectroscopic assay provides new insights about these polymeric proteins.

3-Deoxy-d--oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane. Although labeling of with the chemicalreporter 8-azido-3,8-dideoxy-d--oct-2-ulosonic acid (Kdo-N) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N incorporation into LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an strain lacking endogenous Kdo biosynthesis, supplementation with exogenous Kdo restored full-length core-LPS, which suggests that the Kdo biosynthetic pathways might not be essential in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N-lipid IV and (Kdo-N)-lipid IV by MS analysis. The low level of Kdo-N incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N compared with Kdo. These results indicate that the azido moiety in Kdo-N interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in We propose that our findings will be helpful for researchers using Kdo and its chemical derivatives for investigating LPS biosynthesis, transport, and assembly in Gram-negative bacteria.

Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and "reset" mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases' structure-mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.LpxM | acyltransferase | lipid A | RapidFire mass spectrometry | phosphopantetheine ejection assay

The Gram-negative cell envelope presents a formidable barrier to xenobiotics, and achieving sufficient compound exposure inside the cell is a key challenge for the discovery of new antibiotics. To provide insight on the molecular determinants governing compound exposure in Gram-negative bacteria, we developed a methodology leveraging a cyclooctyne-based bioorthogonal probe to assess compartment-specific compound exposure. This probe can be selectively localized to the periplasmic or cytoplasmic compartments of Gram-negative bacteria. Once localized, the probe is used to test azide-containing compounds for exposure within each compartment by quantifying the formation of click-reaction products by mass spectrometry. We demonstrate this approach is an accurate and sensitive method of determining compartment-specific compound exposure profiles. We then apply this technology to study the compartment-specific exposure profiles of a small panel of azide-bearing compounds with known permeability characteristics in Gram-negative bacteria, demonstrating the utility of the system and the insight it is able to provide regarding compound exposure within intact bacteria.