Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs.

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

The use of NADH anisotropy to investigate mitochondrial cristae alignment.

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340-360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative-especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

Structure of the outer membrane porin OmpW from the pervasive pathogen Klebsiella pneumoniae.

Conjugation is the process by which plasmids, including those that carry antibiotic-resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF-like plasmids relies on the formation of mating-pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid-encoded TraN isoform) and recipient bacteria. Conjugation of the R100-1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid-encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpWKP) is reported at 3.2 Šresolution. OmpWKP forms an eight-stranded ß-barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpWEC) and OmpWKP show high conservation despite sequence variability in the extracellular loops.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Plasmids pick a bacterial partner before committing to conjugation.

Bacterial conjugation was first described by Lederberg and Tatum in the 1940s following the discovery of the F plasmid. During conjugation a plasmid is transferred unidirectionally from one bacterium (the donor) to another (the recipient), in a contact-dependent manner. Conjugation has been regarded as a promiscuous mechanism of DNA transfer, with host range determined by the recipient downstream of plasmid transfer. However, recent data have shown that F-like plasmids, akin to tailed Caudovirales bacteriophages, can pick their host bacteria prior to transfer by expressing one of at least four structurally distinct isoforms of the outer membrane protein TraN, which has evolved to function as a highly sensitive sensor on the donor cell surface. The TraN sensor appears to pick bacterial hosts by binding compatible outer membrane proteins in the recipient. The TraN variants can be divided into specialist and generalist sensors, conferring narrow and broad plasmid host range, respectively. In this review we discuss recent advances in our understanding of the function of the TraN sensor at the donor-recipient interface, used by F-like plasmids to select bacterial hosts within polymicrobial communities prior to DNA transfer.

The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors.

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNß, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNß and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNß-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNß can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, ß, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

Dual-Uptake Mode of the Antibiotic Phazolicin Prevents Resistance Acquisition by Gram-Negative Bacteria.

Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer, Rhizobium sp. strain Pop5. Here, we show that the frequency of spontaneous PHZ-resistant mutants in Sinorhizobium meliloti is below the detection limit. We find that PHZ can enter S. meliloti cells through two distinct promiscuous peptide transporters, BacA and YejABEF, which belong to the SLiPT (SbmA-like peptide transporter) and ABC (ATP-binding cassette) transporter families, respectively. The dual-uptake mode explains the lack of observed resistance acquisition because the simultaneous inactivation of both transporters is necessary for resistance to PHZ. Since both BacA and YejABEF are essential for the development of functional symbiosis of S. meliloti with leguminous plants, the unlikely acquisition of PHZ resistance via the inactivation of these transporters is further disfavored. A whole-genome transposon sequencing screen did not reveal additional genes that can provide strong PHZ resistance when inactivated. However, it was found that the capsular polysaccharide KPS, the novel putative envelope polysaccharide PPP (PHZ-protecting polysaccharide), as well as the peptidoglycan layer jointly contribute to the sensitivity of S. meliloti to PHZ, most likely serving as barriers that reduce the amount of PHZ transported inside the cell. IMPORTANCE Many bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides act either by membrane disruption or by inhibiting essential intracellular processes. The Achilles' heel of the latter type of antimicrobials is their dependence on transporters to enter susceptible cells. Transporter inactivation results in resistance. Here, we show that a rhizobial ribosome-targeting peptide, phazolicin (PHZ), uses two different transporters, BacA and YejABEF, to enter the cells of a symbiotic bacterium, Sinorhizobium meliloti. This dual-entry mode dramatically reduces the probability of the appearance of PHZ-resistant mutants. Since these transporters are also crucial for S. meliloti symbiotic associations with host plants, their inactivation in natural settings is strongly disfavored, making PHZ an attractive lead for the development of biocontrol agents for agriculture.

Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivo S. mansoni. Our crystal structure of the SmVKR2KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target.

Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure.

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae, modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae.

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.

Mating pair stabilization mediates bacterial conjugation species specificity.

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a ß-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNß (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides.

Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo­electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.

Experimental phasing with vanadium and application to nucleotide-binding membrane proteins.

The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein-vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein-vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.

Self-immunity to antibacterial peptides by ABC transporters.

Bacteria produce under certain stress conditions bacteriocins and microcins that display antibacterial activity against closely related species for survival. Bacteriocins and microcins exert their antibacterial activity by either disrupting the membrane or inhibiting essential intracellular processes of the bacterial target. To this end, they can lyse bacterial membranes and cause subsequent loss of their integrity or nutrients, or hijack membrane receptors for internalisation. Both bacteriocins and microcins are ribosomally synthesised and several are posttranslationally modified, whereas others are not. Such peptides are also toxic to the producer bacteria, which utilise immunity proteins or/and dedicated ATP-binding cassette (ABC) transporters to achieve self-immunity and peptide export. In this review, we discuss the structure and mechanism of self-protection that is conferred by these ABC transporters.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

PELDOR/DEER: An Electron Paramagnetic Resonance Method to Study Membrane Proteins in Lipid Bilayers.

Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. This can be achieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complements both X-ray crystallography and cryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.