Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the blind watchmaker of evolution creates skeletal diversity is a key question in contemporary natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug resistant Mycobacterium tuberculosis, and entangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons. less

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element. less

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1 and nucleosomes. We have elucidated the cryo-EM structure of an HP1 dimer bound to an H2A.Z nucleosome, revealing that the HP1 dimer interfaces with nucleosomes at two distinct sites. The primary binding site is located at the N-terminus of histone H3, specifically at the trimethylated K9 (K9me3) region, while a novel secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1 binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. This study offers a model that explains how HP1 functions in heterochromatin maintenance and gene silencing, particularly in the context of H3K9me-dependent mechanisms. Additionally, it sheds light on the H3K9me-independent role of HP1 in responding to DNA damage. less

Background: Invasive plants such as Gorse (Ulex europeaus) may serve as a potentially sustainable source of edible proteins and bioactives through the production of leaf protein concentrates (GLPC). The digestion, uptake and safety of phytochemicals from GLPC and the protein depleted but bioactive rich fraction (PDF) from Gorse was studied using an in vitro simulated digestion model. Results: Both the Gorse fractions and their digesta maintained the viability of cultured (Caco-2) intestinal cells above the 80% threshold, suggesting a lack of overt toxicity. In the digestion model, most of the plant metabolites in the digesta were largely confined to the apical fraction of the Caco-2 monolayer (representing the lumen) and were subsequently expected to be delivered to the colon. Food matrix had a significant but marginal effect on the permeability of metabolites across the Caco-2 monolayer. Lastly, in vitro intestinal microbial action appeared to produce beneficial compounds such as enterolactones, and equol. Conclusion: The Gorse fractions lack general overt toxicity to Caco-2 cells. GLPC and PDF provide metabolic substrates for the colonic microbes to produce secondary compounds that are associated with positive health outcomes. less

The flavivirus NS3 helicase (NS3h), a highly conserved protein, plays a pivotal role in virus replication and thus represents a potential drug target for flavivirus pathogenesis. NS3h utilizes nucleotide triphosphate, such as ATP, for hydrolysis energy (ATPase) to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. The intermediate states along the ATP binding and hydrolysis cycle, as well as the conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. We use extensive molecular dynamics simulations of apo, ATP, ADP+$P_i$, and ADP bound to WNV NS3h+ssRNA to model the conformational ensembles along this cycle. Energetic and structural clustering analyses on these trajectories depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). These findings were experimentally corroborated using viral replicons encoding three mutations at the D471 position. Replication assays using these mutants demonstrated a substantial reduction in viral replication compared to the wild-type. Molecular simulations of the D471 mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open \'valve\' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a \'valve\' for the ATP-pocket, presenting a promising target for antiviral development. less

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) has been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC circadian activity is controlled mainly by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses. less

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp, and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader Replication Factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the E. coli clamp loader at high resolution using cryo-electron microscopy (cryo-EM). We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life. less

Cilia are antenna-like organelles protruding from the surface of many cell types in the human body. Defects in ciliary structure or function often lead to diseases that are collectively called ciliopathies. Cilia and flagella associated protein 410 (CFAP410) localizes at the basal body of cilia/flagella and plays essential roles in ciliogenesis, neuronal development and DNA damage repair. It remains unknown how its specific basal body location is achieved. Multiple single amino acid mutations in CFAP410 have been identified in patients with various ciliopathies. One of the mutations, L224P, is located in the C-terminal domain (CTD) of human CFAP410 and causes severe spondylometaphyseal dysplasia, axial (SMDAX). However, the molecular mechanism for how the mutation causes the disorder remains unclear. Here, we report our structural studies on the CTD of CFAP410 from three distantly related organisms, Homo sapiens, Trypanosoma brucei, and Chlamydomonas reinhardtii. The crystal structures reveal that the three proteins all adopt the same conformation as a tetrameric helical bundle. Our work further demonstrates that the tetrameric assembly of the CTD is essential for the correct localization of CFAP410 in T. brucei, as the L224P mutation that disassembles the tetramer disrupts its basal body localization. Taken together, our studies reveal that the basal body localization of CFAP410 is controlled by the CTD and provide a mechanistic explanation for how the mutation L224P in CFAP410 causes ciliopathies in humans. less

Human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces found on opposite sides of its ACID domain, and referenced as H1 and H2. Here, we use and compare deep learning methods to characterize Human MED25-TADs interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes. Despite these limitations, we experimentally assess the validity of Lana-1 and IE62 MED25 interface predictions. AlphaFold predictions also suggest the existence of a unique hydrophobic pocket for Arabidopsis MED25 ACID domain. less

The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique mode of action. Biosynthetic studies have revealed that darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP). During maturation, the darobactin precursor peptide (DarA) is modified by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) to contain ether and C-C crosslinks. In this work, we describe the enzymatic tolerance of DarE using a panel of DarA variants, revealing that DarE can install the ether and C-C crosslinks independently and in different locations on DarA. These efforts produced 57 darobactin variants, 50 of which were enzymatically modified. Several new variants with fused bicyclic structures were characterized, including darobactin W3Y, which replaces tryptophan with tyrosine at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. Three additional darobactin variants contained fused diether macrocycles, leading us to investigate the origin of ether versus C-C crosslink formation. Computational analyses found that more stable and long-lived C{beta} radicals found on aromatic amino acids correlated with ether formation. Further, molecular docking and calculated transition state structures provide support for the different indole connectivity observed for ether (Trp-C7) and C-C (Trp-C6) crosslink formation. We also provide experimental evidence for a {beta}-oxotryptophan modification, a proposed intermediate during ether crosslink formation. Finally, mutational analysis of the DarA leader region and protein structural predictions identified which residues were dispensable for processing and others that govern substrate engagement by DarE. Our work informs on darobactin scaffold engineering and sheds additional light on the underlying principles of rSAM catalysis. less