Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

High-Resolution Structural Proteomics of Mitochondria Using the 'Build and Retrieve' Methodology.

The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simultaneously identify and solve high-resolution structures of nine essential mitochondrial enzymes with key metabolic functions, including fatty acid catabolism, reactive oxidative species clearance, and amino acid metabolism. Our methodology also identified multiple distinct members of the acyl-CoA dehydrogenase family. This work highlights the potential of cryo-EM to explore tissue proteomics at the atomic level.

A 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins.

Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.

Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport.

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

Structural and Functional Diversity of Resistance-Nodulation-Cell Division Transporters.

Multidrug resistant (MDR) bacteria are a global threat with many common infections becoming increasingly difficult to eliminate. While significant effort has gone into the development of potent biocides, the effectiveness of many first-line antibiotics has been diminished due to adaptive resistance mechanisms. Bacterial membrane proteins belonging to the resistance-nodulation-cell division (RND) superfamily play significant roles in mediating bacterial resistance to antimicrobials. They participate in multidrug efflux and cell wall biogenesis to transform bacterial pathogens into "superbugs" that are resistant even to last resort antibiotics. In this review, we summarize the RND superfamily of efflux transporters with a primary focus on the assembly and function of the inner membrane pumps. These pumps are critical for extrusion of antibiotics from the cell as well as the transport of lipid moieties to the outer membrane to establish membrane rigidity and stability. We analyze recently solved structures of bacterial inner membrane efflux pumps as to how they bind and transport their substrates. Our cumulative data indicate that these RND membrane proteins are able to utilize different oligomerization states to achieve particular activities, including forming MDR pumps and cell wall remodeling machineries, to ensure bacterial survival. This mechanistic insight, combined with simulated docking techniques, allows for the design and optimization of new efflux pump inhibitors to more effectively treat infections that today are difficult or impossible to cure.

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5'-YAG-3' and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1.

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3' acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.

Mapping Protein-Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome-Nascent Globin Complex.

Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein-ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.

Cryo-EM Structures of AcrD Illuminate a Mechanism for Capturing Aminoglycosides from Its Central Cavity.

The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance to aminoglycoside-based drugs by actively extruding these noxious compounds out of E. coli cells. AcrD can also mediate resistance to a limited range of other amphiphilic agents, including bile acids, novobiocin, and fusidic acids. As there is no structural information available for any aminoglycoside-specific RND pump, here we describe cryo-electron microscopy (cryo-EM) structures of AcrD in the absence and presence of bound gentamicin. These structures provide new information about the RND superfamily of efflux pumps, specifically, that three negatively charged residues central to the aminoglycoside-binding site are located within the ceiling of the central cavity of the AcrD trimer. Thus, it is likely that AcrD is capable of picking up aminoglycosides via this central cavity. Through the combination of cryo-EM structural determination, mutagenesis analysis, and molecular simulation, we show that charged residues are critically important for this pump to shuttle drugs directly from the central cavity to the funnel of the AcrD trimer for extrusion. IMPORTANCE Here, we report cryo-EM structures of the AcrD aminoglycoside efflux pump in the absence and presence of bound gentamicin, posing the possibility that this pump is capable of capturing aminoglycosides from the central cavity of the AcrD trimer. The results indicate that AcrD utilizes charged residues to bind and export drugs, mediating resistance to these antibiotics.

Cryo-EM Structures of the Klebsiella pneumoniae AcrB Multidrug Efflux Pump.

The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, ß-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

A cryo-electron microscopic approach to elucidate protein structures from human brain microsomes.

We recently developed a "Build and Retrieve" cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM maps for several individual proteins from a heterogeneous, multiprotein sample. Here we report the use of "Build and Retrieve" to define the composition of a raw human brain microsomal lysate. From this sample, we simultaneously identify and solve cryo-EM structures of five different brain enzymes whose functions affect neurotransmitter recycling, iron metabolism, glycolysis, axonal development, energy homeostasis, and retinoic acid biosynthesis. Interestingly, malfunction of these important proteins has been directly linked to several neurodegenerative disorders, such as Alzheimer's, Huntington's, and Parkinson's diseases. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.

Cryo-Electron Microscopy Structures of a Campylobacter Multidrug Efflux Pump Reveal a Novel Mechanism of Drug Recognition and Resistance.

Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. IMPORTANCE Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Toward structural-omics of the bovine retinal pigment epithelium.

The use of an integrated systems biology approach to investigate tissues and organs has been thought to be impracticable in the field of structural biology, where the techniques mainly focus on determining the structure of a particular biomacromolecule of interest. Here, we report the use of cryoelectron microscopy (cryo-EM) to define the composition of a raw bovine retinal pigment epithelium (RPE) lysate. From this sample, we simultaneously identify and solve cryo-EM structures of seven different RPE enzymes whose functions affect neurotransmitter recycling, iron metabolism, gluconeogenesis, glycolysis, axonal development, and energy homeostasis. Interestingly, dysfunction of these important proteins has been directly linked to several neurodegenerative disorders, including Huntington's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, and schizophrenia. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.

An Analysis of the Novel Fluorocycline TP-6076 Bound to Both the Ribosome and Multidrug Efflux Pump AdeJ from Acinetobacter baumannii.

Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a number of resistance determinants to evade current antibiotics. One of the major resistance mechanisms employed by these pathogens is the use of multidrug efflux pumps. These pumps extrude xenobiotics directly out of bacterial cells, resulting in treatment failures when common antibiotics are administered. Here, the structure of the novel tetracycline antibiotic TP-6076, bound to both the Acinetobacter drug efflux pump AdeJ and the ribosome from Acinetobacter baumannii, using single-particle cryo-electron microscopy (cryo-EM), is elucidated. In this work, the structure of the AdeJ-TP-6076 complex is solved, and we show that AdeJ utilizes a network of hydrophobic interactions to recognize this fluorocycline. Concomitant with this, we elucidate three structures of TP-6076 bound to the A. baumannii ribosome and determine that its binding is stabilized largely by electrostatic interactions. We then compare the differences in binding modes between TP-6076 and the related tetracycline antibiotic eravacycline in both targets. These differences suggest that modifications to the tetracycline core may be able to alter AdeJ binding while maintaining interactions with the ribosome. Together, this work highlights how different mechanisms are used to stabilize the binding of tetracycline-based compounds to unique bacterial targets and provides guidance for the future clinical development of tetracycline antibiotics. IMPORTANCE Treatment of antibiotic-resistant organisms such as A. baumannii represents an ongoing issue for modern medicine. The multidrug efflux pump AdeJ serves as a major resistance determinant in A. baumannii through its action of extruding antibiotics from the cell. In this work, we use cryo-EM to show how AdeJ recognizes the experimental tetracycline antibiotic TP-6076 and prevents this drug from interacting with the A. baumannii ribosome. Since AdeJ and the ribosome use different binding modes to stabilize interactions with TP-6076, exploiting these differences may guide future drug development for combating antibiotic-resistant A. baumannii and potentially other strains of MDR bacteria.

Cryo-EM Determination of Eravacycline-Bound Structures of the Ribosome and the Multidrug Efflux Pump AdeJ of Acinetobacter baumannii.

Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the Acinetobacter drug efflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. IMPORTANCE Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.

Cryo-EM as a tool to study bacterial efflux systems and the membrane proteome.

Antibiotic resistance is an emerging threat to global health. Current treatment regimens for these types of bacterial infections are becoming increasingly inadequate. Thus, new innovative technologies are needed to help identify and characterize novel drugs and drug targets which are critical in order to combat multidrug-resistant bacterial strains. Bacterial efflux systems have emerged as an attractive target for drug design, as blocking their export function significantly increases the potency of administered antibiotics. However, in order to develop potent and tolerable efflux pump inhibitors with high efficacy, detailed structural information is required for both the apo- and substrate-bound forms of these membrane proteins. The emergence of cryo-electron microscopy (cryo-EM) has greatly advanced the field of membrane protein structural biology. It has significantly enhanced the ability to solve large multi-protein complexes as well as extract meaningful data from a heterogeneous sample, such as identification of several assembly states of the bacterial ribosome, from a single data set. This technique can be expanded to solve the structures of substrate-bound efflux pumps and entire efflux systems from previously unusable membrane protein sample preparations. Subsequently, cryo-EM combined with other biophysical techniques has the potential to markedly advance the field of membrane protein structural biology. The ability to discern complete transport machineries, enzymatic signal transduction pathways, and other membrane-associated complexes will help us fully understand the complexities of the membrane proteome.

Cryoelectron Microscopy Structures of AdeB Illuminate Mechanisms of Simultaneous Binding and Exporting of Substrates.

Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent Acinetobacterdrug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into "dimer plus monomer" and "monomer plus monomer plus monomer" configurations. We also discover that a single AdeB protomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dynamics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently.IMPORTANCEAcinetobacter baumannii has emerged as one of the most highly antibiotic-resistant Gram-negative pathogens. The prevalent AdeB multidrug efflux pump mediates resistance to a broad spectrum of clinically relevant antimicrobial agents. Here, we report six cryo-EM structures of the trimeric AdeB pump in the presence of ethidium bromide. We discover that a single AdeB protomer can simultaneously anchor a number of ligands, and different AdeB protomers can bind ethidium molecules simultaneously. The results indicate that each AdeB protomer within the trimer recognizes and extrudes drugs independently.

Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development.

Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections.IMPORTANCEAcinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump.

Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle.IMPORTANCEAcinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as ß-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.