Elucidating the dynamics of Integrin αIIb[beta]3 from native platelet membranes by cryo-EM with build and retrieve method.

Platelets fulfill their essential physiological roles sensing the extracellular environment through their membrane proteins. The native membrane environment provides essential regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins to be solved from homogeneous samples.

Cryo-EM structures of the human band 3 transporter indicate a transport mechanism involving the coupled movement of chloride and bicarbonate ions.

The band 3 transporter is a critical integral membrane protein of the red blood cell (RBC), as it is responsible for catalyzing the exchange of bicarbonate and chloride anions across the plasma membrane. To elucidate the structural mechanism of the band 3 transporter, detergent solubilized human ghost membrane reconstituted in nanodiscs was applied to a cryo-EM holey carbon grid to define its composition. With this approach, we identified and determined structural information of the human band 3 transporter.

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.

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

Bacterial 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.

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.

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

Moseng 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.

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.

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.

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.

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.

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.

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.

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

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 is the prevalent rug fflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily.

Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT.

The intra-erythrocyte stage of P. falciparum relies primarily on glycolysis to generate adenosine triphosphate (ATP) and the energy required to support growth and reproduction. Lactic acid, a metabolic byproduct of glycolysis, is potentially toxic as it lowers the pH inside the parasite.

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.

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S.

Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance.

is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in is the ultiple ransferrable esistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g.

Structure and function of LCI1: a plasma membrane CO channel in the Chlamydomonas CO concentrating mechanism.

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C ) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C uptake systems.

Structural and functional evidence that lipoprotein LpqN supports cell envelope biogenesis in .

The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research.

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.

MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine.

The cell envelope of is notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear.

A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load.

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library.

Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump.

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules.