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Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Polyamines, characterized by their polycationic nature, are ubiquitously present in all organisms and play numerous cellular functions. Among polyamines, spermidine stands out as the predominant type in both prokaryotic and eukaryotic cells. The PotD-PotABC protein complex in Escherichia coli , belonging to the adenosine triphosphate-binding cassette transporter family, is a spermidine-preferential uptake system. Here, we report structural details of the polyamine uptake system PotD-PotABC in various states. Our analyses reveal distinct "inward-facing" and "outward-facing" conformations of the PotD-PotABC transporter, as well as conformational changes in the "gating" residues (F222, Y223, D226, and K241 in PotB; Y219 and K223 in PotC) controlling spermidine uptake. Therefore, our structural analysis provides insights into how the PotD-PotABC importer recognizes the substrate-binding protein PotD and elucidates molecular insights into the spermidine uptake mechanism of bacteria.

Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by 1 H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor's biological activity.

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the β-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity., (© 2024. The Author(s).)

Glucose is the primary source of energy for many organisms and is efficiently taken up by bacteria through a dedicated transport system that exhibits high specificity. In Escherichia coli, the glucose-specific transporter IICB Glc serves as the major glucose transporter and functions as a component of the phosphoenolpyruvate-dependent phosphotransferase system. Here, we report cryo-electron microscopy (cryo-EM) structures of the glucose-bound IICB Glc protein. The dimeric transporter embedded in lipid nanodiscs was captured in the occluded, inward- and occluded, outward-facing conformations. Together with biochemical and biophysical analyses, and molecular dynamics (MD) simulations, we provide insights into the molecular basis and dynamics for substrate recognition and binding, including the gates regulating the binding sites and their accessibility. By combination of these findings, we present a mechanism for glucose transport across the plasma membrane. Overall, this work provides molecular insights into the structure, dynamics, and mechanism of the IICB Glc transporter in a native-like lipid environment., (© 2024. The Author(s).)

A series of arylsulfones and heteroarylsulfones have previously been demonstrated to dysregulate the conserved bacterial ClpP protease, causing the unspecific degradation of essential cellular housekeeping proteins and ultimately resulting in cell death. A cocrystal structure of a 2-β-sulfonylamide analog, ACP1-06, with Escherichia coli ClpP showed that its 2-pyridyl sulfonyl substituent adopts two orientations in the binding site related through a sulfone bond rotation. From this, a new bis -aryl phosphine oxide scaffold, designated as ACP6, was designed based on a "conformation merging" approach of the dual orientation of the ACP1-06 sulfone. One analog, ACP6-12, exhibited over a 10-fold increase in activity over the parent ACP1-06 compound, and a cocrystal X-ray structure with ClpP confirmed its predicted binding conformation. This allowed for a comparative analysis of how different ligand classes bind to the hydrophobic binding site. The study highlights the successful application of structure-based rational design of novel phosphine oxide-based antibiotics.

EmrE is a bacterial efflux protein in the small multidrug-resistant (SMR) family present in Escherichia coli . Due to its small size, 110 residues in each dimer subunit, it is an ideal model system to study ligand-protein-membrane interactions. Here in our work, we have calculated the free energy landscape of benzyltrimetylammonium (BTMA) and tetraphenyl phosphonium (TPP) binding to EmrE using the enhanced sampling method-multiple walker metadynamics. We estimate that the free energy of BTMA binding to EmrE is -21.2 ± 3.3 kJ/mol and for TPP is -43.6 ± 3.8 kJ/mol. BTMA passes through two metastable states to reach the binding pocket, while TPP has a more complex binding landscape with four metastable states and one main binding site. Our simulations show that the ligands interact with the membrane lipids at a distance 1 nm away from the binding site which forms a broad local minimum, consistent for both BTMA and TPP. This site can be an alternate entry point for ligands to partition from the membrane into the protein, especially for bulky and/or branched ligands. We also observed the membrane lipid and C-terminal 110HisA form salt-bridge interactions with the helix-1 residue 22LysB. Our free energy estimates and clusters are in close agreement with experimental data and give us an atomistic view of the ligand-protein-lipid interactions. Understanding the binding pathway of these ligands can guide us in future design of ligands that can alter or halt the function of EmrE.

π-Stacking, a type of noncovalent interactions involving aromatic residues, plays an important role in protein folding and function. In this work, an attempt has been made to measure CH/π and NH/π stacking interactions in a protein in Escherichia coli cells using a combined double-mutant cycle and nuclear magnetic resonance spectroscopy method. The results show that the CH/π and NH/π stacking interactions are generally weaker in cells than those in the buffer. The transient intermolecular noncovalent interactions between the protein and the complex cellular environment may compete with and thus weaken the stacking interactions in the protein. The weakening of stacking interactions can enhance the local conformational opening of proteins in E. coli cells. This is evident from the faster rates of amide hydrogen/deuterium exchange observed in cells than in the buffer, for residues that undergo local conformational opening. This study highlights the influence of the cellular environment on π-stacking and the conformational dynamics of proteins.

The Rcs pathway is repressed by the inner membrane protein IgaA under non-stressed conditions. This repression is hypothesized to be relieved by the binding of the outer membrane-anchored RcsF to IgaA. However, the precise mechanism by which RcsF binding triggers the signaling remains unclear. Here, we present the 1.8 Å resolution crystal structure capturing the interaction between IgaA and RcsF. Our comparative structural analysis, examining both the bound and unbound states of the periplasmic domain of IgaA (IgaAp), highlights rotational flexibility within IgaAp. Conversely, the conformation of RcsF remains unchanged upon binding. Our in vivo and in vitro studies do not support the model of a stable complex involving RcsF, IgaAp, and RcsDp. Instead, we demonstrate that the elements beyond IgaAp play a role in the interaction between IgaA and RcsD. These findings collectively allow us to propose a potential mechanism for the signaling across the inner membrane through IgaA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B 12 transport protein BtuB by specifically binding coenzyme B 12 (AdoCbl) as its natural ligand. The B 12 sensing riboswitch class is known to accept various B 12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B 12 show larger effects indicating crucial B 12 -RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B 12 -btuB riboswitch recognition clearly beyond the known role in affinity., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis., (© 2024. The Author(s).)

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding 1-3 . The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES 4-6 . To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells., (© 2024. The Author(s).)

Tuberculosis (TB) remains a critical global health challenge, with the emergence of drug-resistant strains heightening concerns. The development of effective drugs targeting both wild-type (WT) and mutant Escherichia coli RNA polymerase β subunit (RpoB) is crucial for global TB control, aiming to alleviate TB incidence, mortality, and transmission. This study employs molecular docking and ADMET analyses to screen echinoderm metabolites for their potential inhibition of Escherichia coli RNA polymerase, focusing on wild-type and mutant RpoB variants associated with TB drug resistance. The evaluation of docking results using the glide gscore led to the selection of the top 10 compounds for each protein receptor. Notably, CMNPD2176 demonstrated the highest binding affinity against wild-type RpoB, CMNPD13873 against RpoB D516V mutant, CMNPD2177 against RpoB H526Y mutant, and CMNPD11620 against RpoB S531L mutant. ADMET screening confirmed the therapeutic potential of these selected compounds. Additionally, MM-GBSA binding free energy calculations and molecular dynamics simulations provided further support for the docking investigations. While the results suggest these compounds could be viable for tuberculosis treatment, it is crucial to note that further in-vitro research is essential for the transition from prospective inhibitors to clinical drugs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alsulais et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Polymers can facilitate detergent-free extraction of membrane proteins into nanodiscs (e.g., SMALPs, DIBMALPs), incorporating both integral membrane proteins as well as co-extracted native membrane lipids. Lipid-only SMALPs and DIBMALPs have been shown to possess a unique property; the ability to exchange lipids through 'collisional lipid mixing'. Here we expand upon this mixing to include protein-containing DIBMALPs, using the rhomboid protease GlpG. Through lipidomic analysis before and after incubation with DMPC or POPC DIBMALPs, we show that lipids are rapidly exchanged between protein and lipid-only DIBMALPs, and can be used to identify bound or associated lipids through 'washing-in' exogenous lipids. Additionally, through the requirement of rhomboid proteases to cleave intramembrane substrates, we show that this mixing can be performed for two protein-containing DIBMALP populations, assessing the native function of intramembrane proteolysis and demonstrating that this mixing has no deleterious effects on protein stability or structure., (© 2024. The Author(s).)

Iron-sulfur (Fe-S) clusters are ubiquitous and are necessary to sustain basic life processes. The intracellular Fe-S clusters do not form spontaneously and many proteins are required for their biosynthesis and delivery. The bacterial P-loop NTPase family protein ApbC participates in Fe-S cluster assembly and transfers the cluster into apoproteins, with the Walker A motif and CxxC motif being essential for functionality of ApbC in Fe-S protein biogenesis. However, the structural basis underlying the ApbC activity and the motifs' role remains unclear. Here, we report the crystal structure of Escherichia coli ApbC at 2.8 Å resolution. The dimeric structure is in a W shape and the active site is located in the 2-fold center. The function of the motifs can be annotated by structural analyses. ApbC has an additional N-terminal domain that differs from other P-loop NTPases, possibly conferring its inherent specificity in vivo., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Efflux-mediated β-lactam resistance is a major public health concern, reducing the effectiveness of β-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines β-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under β-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms., (© 2024. The Author(s).)

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors., Competing Interests: Competing interests statement:The authors declare no competing interest.

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a β-roll upon binding the ops -paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops -paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the β3/β4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15 N backbone chemical shift differences are associated with the β3/β4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.

Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an "overcompensation" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented., Competing Interests: Competing interests statement:The authors declare no competing interest.

To enable an efficient bacterial cell surface display with effective protein expression and cell surface loading ability via autotransporter for potential vaccine development applications, the inner membrane protein translocation efficiency was investigated via a trial-and-error strategy by replacing the original unusual long signal peptide of E. coli Ag43 with 11 different signal peptides. The receptor-binding domain (RBD) of coronavirus was used as a neutral display substrate to optimize the expression conditions, and the results showed that signal peptides from PelB, OmpC, OmpF, and PhoA protein enhance the bacterial cell surface display efficiency of RBD. In addition, the temperature has also a significant effect on the autodisplay efficiency of RBD. Our data provide further technical basis for the biotechnological application of Ag43 as a bacterial surface display carrier system and further potential application in vaccine development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method - Ni-affinity chromatography using a C-terminal poly-histidine tag - altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.

Nanoelectromechanical systems (NEMS)-based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real-time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single-molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 μm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS-MS shows good agreement with the known mass of GroEL., (© 2024 Wiley-VCH GmbH.)

Transporter proteins carry their substrate across the cell membrane by changing their conformation. Thus, conformational dynamics are crucial for transport function. However, clarifying the complete transport cycle is challenging even with the current structural biology approach. Molecular dynamics (MD) simulation is a computational approach that can provide the time-resolved conformational dynamics of transporter proteins in atomic details but suffers from a high computational cost. Here, we integrate state-of-the-art protein structure prediction AI, AlphaFold2 (AF2), with MD simulation to reduce the computational cost. Focusing on the transporter protein NarK, we first show that AF2 sampled broad conformations of NarK, including the inward-open, occluded, and outward-open states. We also applied the coevolution-informed mutation in AF2, identifying state-shifting mutations. Then, we show that MD simulations from AF2-generated outward-open conformation, which is experimentally unresolved, captured the essence of the conformational state. We also found that MD simulations from AF2-generated intermediates showed transient dynamics like a transition state connecting two conformational states. This study paves the way for efficient conformational sampling of transporter proteins.

Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.

Fragmentation trends of large peptides were characterized by five activation methods, including HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD. Sequence coverages and scores were assessed based on charge site, peptide sequence, and peptide size. The effect of charge state and peptide size on sequence coverage was explored for a Glu-C digest of E. coli ribosomal proteins, and linear regression analysis of the collection of peptides indicated that HCD, ETD, and EThcD have a higher dependence charge state than 193 and 213 nm UV. Four model peptides, neuromedin, glucagon, galanin, and amyloid β, were characterized in greater detail based on charge site analysis and showed a charge state dependence on sequence coverage for collision and electron-based activation methods.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives., Competing Interests: Competing interests statement:The authors declare no competing interest.

Antigen 43 (Ag43) proteins, found on the outer membrane of Escherichia coli, are β-sheets that fold into a unique cylindrical structure known as a β-barrel. There are several known structural similarities between bacterial Ag43 autotransporters and physical components; however, the factors that stabilize the barrel and the mechanism for Ag43 passenger domainmediated translocation across the pore of the β-barrel remain unclear. In this study, we analyzed Ag43β-enhanced green fluorescent protein chimeric variants to provide new insights into the autotransporter Ag43β-barrel assembly, focusing on the impact of the α-helical linker domain. Among the chimeric variants, Ag43β700 showed the highest surface display, which was confirmed through extracellular protease digestion, flow cytometry, and an evaluation of outer membrane vesicles (OMVs). The Ag43β700 module offered reliable information on stable barrel folding and chimera expression at the exterior of the OMVs. [BMB Reports 2024; 57(8): 369-374].

Cytochrome bo 3 quinol oxidase belongs to the heme‑copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo 3 has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo 3 from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1-41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a "U-shape" assembly in contrast to the transmembrane helix, named "TM0", in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo 3 conformations should exist. Our structural model presents experimental evidence for an "open" conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions., (© 2024. The Author(s).)

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the β-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

Enterobactin is a high-affinity iron chelator produced and secreted by Escherichia coli and Salmonella typhimurium to scavenge scarce extracellular Fe 3+ as a micronutrient. EntC and EntB are the first two enzymes in the enterobactin biosynthetic pathway. Isochorismate, produced by EntC, is a substrate for EntB isochorismatase. By using a competing isochorismate-consuming enzyme (the E. coli SEPHCHC synthase MenD), we found in a coupled assay that residual EntB isochorismatase activity decreased as a function of increasing MenD concentration. In the presence of excess MenD, EntB isochorismatase activity was observed to decrease by 84%, indicative of partial EntC-EntB channeling (16%) of isochorismate. Furthermore, addition of glycerol to the assay resulted in an increase of residual EntB isochorismatase activity to approximately 25% while in the presence of excess MenD. These experimental outcomes supported the existence of a substrate channeling surface identified in a previously reported protein-docking model of the EntC-EntB complex. Two positively charged EntB residues (K21 and R196) that were predicted to electrostatically guide negatively charged isochorismate between the EntC and EntB active sites were mutagenized to determine their effects on substrate channeling. The EntB variants K21D and R196D exhibited a near complete loss of isochorismatase activity, likely due to electrostatic repulsion of the negatively charged isochorismate substrate. Variants K21A, R196A, and K21A/R196A retained partial EntB isochorismatase activity in the absence of EntC; in the presence of EntC, isochorismatase activity in all variants increased to near wild-type levels. The MenD competition assay of the variants revealed that while K21A channeled isochorismate as efficiently as wild-type EntB (~ 15%), the variants K21A/R196A and R196A exhibited an approximately 5-fold loss in observed channeling efficiency (~3%). Taken together, these results demonstrate that partial substrate channeling occurs between EntC and EntB via a leaky electrostatic tunnel formed upon dynamic EntC-EntB complex formation and that EntB R196 plays an essential role in isochorismate channeling., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions., (© 2024 Wiley‐VCH GmbH.)

High-speed single-molecule tracking in live cells is becoming an increasingly popular method for quantifying the spatiotemporal behavior of proteins in vivo . The method provides a wealth of quantitative information, but users need to be aware of biases that can skew estimates of molecular mobilities. The range of suitable fluorophores for live-cell single-molecule imaging has grown substantially over the past few years, but it remains unclear to what extent differences in photophysical properties introduce biases. Here, we tested two fluorophores with entirely different photophysical properties, one that photoswitches frequently between bright and dark states (TMR) and one that shows exceptional photostability without photoswitching (JFX650). We used a fusion of the Escherichia coli DNA repair enzyme MutS to the HaloTag and optimized sample preparation and imaging conditions for both types of fluorophore. We then assessed the reliability of two common data analysis algorithms, mean-square displacement (MSD) analysis and Hidden Markov Modeling (HMM), to estimate the diffusion coefficients and fractions of MutS molecules in different states of motion. We introduce a simple approach that removes discrepancies in the data analyses and show that both algorithms yield consistent results, regardless of the fluorophore used. Nevertheless, each dye has its own strengths and weaknesses, with TMR being more suitable for sampling the diffusive behavior of many molecules, while JFX650 enables prolonged observation of only a few molecules per cell. These characterizations and recommendations should help to standardize measurements for increased reproducibility and comparability across studies.

We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis -sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium . A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.

S -Adenosylmethionine (SAM) is a crucial metabolic intermediate playing irreplaceable roles in organismal activities. However, the synthesis of SAM by methionine adenosyltransferase (MAT) is hindered by low conversion due to severe product inhibition. Herein structure-guided semirational engineering was conducted on MAT from Escherichia coli ( Ec MAT) to mitigate the product inhibitory effect. Compared with the wild-type Ec MAT, the best variant E56Q/Q105R exhibited an 8.13-fold increase in half maximal inhibitory concentration and a 4.46-fold increase in conversion (150 mM ATP and l-methionine), leading to a SAM titer of 47.02 g/L. Another variant, E56N/Q105R, showed superior thermostability with an impressive 85.30-fold increase in half-life (50 °C) value. Furthermore, molecular dynamics (MD) simulation results demonstrate that the alleviation in product inhibitory effect could be attributed to facilitated product release. This study offers molecular insights into the mitigated product inhibition, and provides valuable guidance for engineering MAT toward enhanced catalytic performance.

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD., (© 2024. The Author(s).)

Bacteria rely on two-component systems to sense environmental cues and regulate gene expression for adaptation. The PhoQ/PhoP system exemplifies this crucial role, playing a key part in sensing magnesium (Mg 2+ ) levels, antimicrobial peptides, mild acidic pH, osmotic upshift, and long-chain unsaturated fatty acids, promoting virulence in certain bacterial species. However, the precise details of PhoQ activation remain elusive. To elucidate PhoQ's signaling mechanism at atomic resolution, we combined AlphaFold2 predictions with molecular modeling and carried out extensive Molecular Dynamics (MD) simulations. Our MD simulations revealed three distinct PhoQ conformations that were validated by experimental data. Notably, one conformation was characterized by Mg 2+ bridging the acidic patch in the sensor domain to the membrane, potentially representing a repressed state. Furthermore, the high hydration observed in a putative intermediate state lends support to the hypothesis of water-mediated conformational changes during PhoQ signaling. Our findings not only revealed specific conformations within the PhoQ signaling pathway, but also hold significant promise for understanding the broader histidine kinase family due to their shared structural features. Our approach paves the way for a more comprehensive understanding of histidine kinase signaling mechanisms across various bacterial species and opens the door for developing novel therapeutics that target PhoQ modulation., (© 2024. The Author(s).)

Protein solubility plays a crucial role in various biotechnological, industrial, and biomedical applications. With the reduction in sequencing and gene synthesis costs, the adoption of high-throughput experimental screening coupled with tailored bioinformatic prediction has witnessed a rapidly growing trend for the development of novel functional enzymes of interest (EOI). High protein solubility rates are essential in this process and accurate prediction of solubility is a challenging task. As deep learning technology continues to evolve, attention-based protein language models (PLMs) can extract intrinsic information from protein sequences to a greater extent. Leveraging these models along with the increasing availability of protein solubility data inferred from structural database like the Protein Data Bank holds great potential to enhance the prediction of protein solubility. In this study, we curated an Updated Escherichia coli protein Solubility DataSet (UESolDS) and employed a combination of multiple PLMs and classification layers to predict protein solubility. The resulting best-performing model, named Protein Language Model-based protein Solubility prediction model (PLM_Sol), demonstrated significant improvements over previous reported models, achieving a notable 6.4% increase in accuracy, 9.0% increase in F1_score, and 11.1% increase in Matthews correlation coefficient score on the independent test set. Moreover, additional evaluation utilizing our in-house synthesized protein resource as test data, encompassing diverse types of enzymes, also showcased the good performance of PLM_Sol. Overall, PLM_Sol exhibited consistent and promising performance across both independent test set and experimental set, thereby making it well suited for facilitating large-scale EOI studies. PLM_Sol is available as a standalone program and as an easy-to-use model at https://zenodo.org/doi/10.5281/zenodo.10675340., (© The Author(s) 2024. Published by Oxford University Press.)

Ribosome biogenesis is a highly regulated cellular process that involves the control of numerous assembly factors. The small protein YjgA has been reported to play a role in the late stages of 50S assembly. However, the precise molecular mechanism underlying its function remains unclear. In this study, cryo-electron microscopy (cryo-EM) structures revealed that depletion of YjgA or its N-terminal loop in Escherichia coli both lead to the accumulation of immature 50S particles with structural abnormalities mainly in peptidyl transferase center (PTC) and H68/69 region. CryoDRGN analysis uncovered 8 and 6 distinct conformations of pre50S for ΔyjgA and YjgA-ΔNloop, respectively. These conformations highlighted the role of the N-terminal loop of YjgA in integrating uL16 and stabilizing H89 in PTC, which was further verified by the pull-down assays of YjgA and its mutants with uL16. Together with the function of undocking H68 through the binding of its C-terminal CTLH-like domain to the base of the L1 stalk, YjgA facilitates the maturation of PTC. This study identified critical domains of YjgA contributing to 50S assembly efficiency, providing a comprehensive understanding of the dual roles of YjgA in accelerating ribosome biogenesis and expanding our knowledge of the intricate processes governing cellular protein synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC 50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies., Competing Interests: The authors declare no conflict of interest.

Colanic acid (CA) is exopolysaccharide that presents growing potential in the food and healthcare industry as a versatile polymer. Previously, we have constructed the Escherichia coli strain WWM16 which can efficiently produce CA. In this study, WWM16 has been further engineered to produce a higher yield of CA with low molecular mass and viscosity. The gene mcbR encoding a transcriptional factor, and the genes opgD, opgG , and opgH related to the biosynthesis of osmoregulated periplasmic glucans were deleted in E. coli WWM16, and the resulting strain WWM166 produced 18.1 g/L CA. The expression level of wcaD encoding the polymerase in WWM166 was downregulated using CRISPRi. As a result, the strain WWM166/pWpD1 could produce 49.9 g/L CA with lower molecular mass. CA products were purified from both WWM166 and WWM166/pWpD1, and their molecular mass, viscosity, fluidity, hygroscopicity, and antioxidant activity were determined and compared. These findings demonstrate the potential application of CA with different molecular masses to prolong life and protect skin in the food and cosmetic industries.

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features., Competing Interests: Competing interests statement:J.M.C., A.H.L., I.M.D., H.B., and J.H.G. hold US patent 10,359,395 on the Nanopore Tweezers technology.

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein life cycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favorable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpy-driven, with micromolar affinity, while nanomolar affinity is achieved through a favorable entropic contribution for both intrinsically disordered and folding-competent nascent chains. These findings suggest a general mechanism for cotranslational TF function, which relies on occupation of the exposed TF-substrate binding groove rather than specific complementarity between chaperone and nascent chain. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

In this issue of Molecular Cell, Roeselová et al. 1 provide insights into co-translational folding of a multidomain protein in bacteria, revealing how the chaperones Trigger Factor, DnaJ, and DnaK work together to facilitate the folding of nascent chains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The acyl carrier protein of Escherichia coli , termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP's hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP's thioester linkages with 15 N-labeled amide bonds, which facilitated direct "visualization" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP's hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.