Structural analysis of resistance-nodulation cell division transporters.

SUMMARYInfectious bacteria have both intrinsic and acquired mechanisms to combat harmful biocides that enter the cell. Through adaptive pressures, many of these pathogens have become resistant to many, if not all, of the current antibiotics used today to treat these often deadly infections. One prominent mechanism is the upregulation of efflux systems, especially the resistance-nodulation-cell division class of exporters. These tripartite systems consist of an inner membrane transporter coupled with a periplasmic adaptor protein and an outer membrane channel to efficiently transport a diverse array of substrates from inside the cell to the extracellular space. Detailed mechanistic insight into how these inner membrane transporters recognize and shuttle their substrates can ultimately inform both new antibiotic and efflux pump inhibitor design. This review examines the structural basis of substrate recognition of these pumps and the molecular mechanisms underlying multidrug extrusion, which in turn mediate antimicrobial resistance in bacterial pathogens.

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.

Structural basis of DNA recognition of the Campylobacter jejuni CosR regulator.

Campylobacter jejuni is a foodborne pathogen commonly found in the intestinal tracts of animals. This pathogen is a leading cause of gastroenteritis in humans. Besides its highly infectious nature, C. jejuni is increasingly resistant to a number of clinically administrated antibiotics. As a consequence, the Centers for Disease Control and Prevention has designated antibiotic-resistant Campylobacter as a serious antibiotic resistance threat in the United States. The C. jejuni CosR regulator is essential to the viability of this bacterium and is responsible for regulating the expression of a number of oxidative stress defense enzymes. Importantly, it also modulates the expression of the CmeABC multidrug efflux system, the most predominant and clinically important system in C. jejuni that mediates resistance to multiple antimicrobials. Here, we report structures of apo-CosR and CosR bound with a 21 bp DNA sequence located at the cmeABC promotor region using both single-particle cryo-electron microscopy and X-ray crystallography. These structures allow us to propose a novel mechanism for CosR regulation that involves a long-distance conformational coupling and rearrangement of the secondary structural elements of the regulator to bind target DNA. IMPORTANCE: Campylobacter jejuni has emerged as an antibiotic-resistant threat worldwide. CosR is an essential regulator for this bacterium and is important for Campylobacter adaptation to various stresses. Here, we describe the structural basis of CosR binding to target DNA as determined by cryo-electron microscopy and X-ray crystallography. Since CosR is a potential target for intervention, our studies may facilitate the development of novel therapeutics to combat C. jejuni infection.

Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1.

Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.

Bacterial efflux pump modulators prevent bacterial growth in macrophages and under broth conditions that mimic the host environment.

New approaches for combating microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen, Salmonella enterica serotype Typhimurium (S. Typhimurium), in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy. Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome, acrAB is required for growth, the EPM analogs are bacteriostatic, and the EPM analogs increase the potency of antibiotics. These data suggest that under macrophage-like conditions, the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics.IMPORTANCEBacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics.

Bacterial Efflux Pump Modulators Prevent Bacterial Growth in Macrophages and Under Broth Conditions that Mimic the Host Environment.

New approaches for combatting microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division (RND) family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen, Salmonella enterica serotype Typhimurium (S. Typhimurium) in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy (cryo-EM). Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome, acrAB is required for growth, the EPM analogs are bacteriostatic, and increase the potency of antibiotics. These data suggest that under macrophage-like conditions the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics.

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.

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.

Response to Comment on "Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling".

Moseng et al. recently reported four cryo-electron microscopy structures of the human Na-K-2Cl cotransporter-1 (hNKCC1), both in the absence and presence of bound loop diuretic (furosemide or bumetanide). This research article included high-resolution structural information for a previously undefined structure of apo-hNKCC1 containing both the transmembrane and cytosolic carboxyl-terminal domains. The manuscript also demonstrated various conformational states of this cotransporter induced by diuretic drugs. On the basis of the structural information, the authors proposed a scissor-like inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1. This work has provided important insights into the mechanism of inhibition and substantiated the concept of a long-distance coupling involving movements of both the transmembrane and carboxyl-terminal cytoplasmic domains for inhibition.

High-resolution structural-omics of human liver enzymes.

We applied raw human liver microsome lysate to a holey carbon grid and used cryo-electron microscopy (cryo-EM) to define its composition. From this sample we identified and simultaneously determined high-resolution structural information for ten unique human liver enzymes involved in diverse cellular processes. Notably, we determined the structure of the endoplasmic bifunctional protein H6PD, where the N- and C-terminal domains independently possess glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase enzymatic activity, respectively. We also obtained the structure of heterodimeric human GANAB, an ER glycoprotein quality-control machinery that contains a catalytic α subunit and a noncatalytic ß subunit. In addition, we observed a decameric peroxidase, PRDX4, which directly contacts a disulfide isomerase-related protein, ERp46. Structural data suggest that several glycosylations, bound endogenous compounds, and ions associate with these human liver enzymes. These results highlight the importance of cryo-EM in facilitating the elucidation of human 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.

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.

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.

RND Pump-Mediated Efflux of Amotosalen, a Compound Used in Pathogen Inactivation Technology to Enhance Safety of Blood Transfusion Products, May Compromise Its Gram-Negative Anti-Bacterial Activity.

Pathogen inactivation is a strategy to improve the safety of transfusion products. The only pathogen reduction technology for blood products currently approved in the US utilizes a psoralen compound, called amotosalen, in combination with UVA light to inactivate bacteria, viruses, and protozoa. Psoralens have structural similarity to bacterial multidrug efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through genetic, biophysical, and molecular modeling analysis. The main efflux systems in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa are tripartite resistance-nodulation-cell division (RND) systems, which span the inner and outer membranes of Gram-negative pathogens, and expel antibiotics from the bacterial cytoplasm into the extracellular space. We provide evidence that amotosalen is an efflux substrate for the E. coli AcrAB, Acinetobacter baumannii AdeABC, and P. aeruginosa MexXY RND efflux pumps. Furthermore, we show that the MICs for contemporary Gram-negative bacterial isolates for these species and others in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens. IMPORTANCE Pathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by major efflux pumps in E. coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the MICs for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

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.

Simultaneous solving high-resolution structures of various enzymes from human kidney microsomes.

The ability to investigate tissues and organs through an integrated systems biology approach has been thought to be unobtainable in the field of structural biology, where the techniques mainly focus on a particular biomacromolecule of interest. Here we report the use of cryo-electron microscopy (cryo-EM) to define the composition of a raw human kidney microsomal lysate. We simultaneously identify and solve cryo-EM structures of four distinct kidney enzymes whose functions have been linked to protein biosynthesis and quality control, biosynthesis of retinoic acid, gluconeogenesis and glycolysis, and the regulation and metabolism of amino acids. Interestingly, all four of these enzymes are directly linked to cellular processes that, when disrupted, can contribute to the onset and progression of diabetes. This work underscores the potential of cryo-EM to facilitate tissue and organ proteomics at the atomic level.

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.

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.

Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling.

The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na+-dependent transporter responsible for simultaneously translocating Na+, K+, and Cl- ions into cells. In human tissue, NKCC1 plays a critical role in regulating cytoplasmic volume, fluid intake, chloride homeostasis, and cell polarity. Here, we report four structures of human NKCC1 (hNKCC1), both in the absence and presence of loop diuretic (bumetanide or furosemide), using single-particle cryo-electron microscopy. These structures allow us to directly observe various novel conformations of the hNKCC1 dimer. They also reveal two drug-binding sites located at the transmembrane and cytosolic carboxyl-terminal domains, respectively. Together, our findings enable us to delineate an inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.