Allosteric activation of preformed EGF receptor dimers by a single ligand binding event.

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor in vivo. Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Antibody Complexes.

Monoclonal based therapeutics have always been looked at as a futuristic natural way we could take care of pathogens and many diseases. However, in order to develop, establish and realize monoclonal based therapy we need to understand how the immune system contains or kill pathogens. Antibody complexes serve the means to decode this black box. We have discussed examples of antibody complexes both at biochemical and structural levels to understand and appreciate how discoveries in the field of antibody complexes have started to decoded mechanism of viral invasion and create potential vaccine targets against many pathogens. Antibody complexes have made advancement in our knowledge about the molecular interaction between antibody and antigen. It has also led to identification of potent protective monoclonal antibodies. Further use of selective combination of monoclonal antibodies have provided improved protection against deadly diseases. The administration of newly designed and improved immunogen has been used as potential vaccine. Therefore, antibody complexes are important tools to develop new vaccine targets and design an improved combination of monoclonal antibodies for passive immunization or protection with very little or no side effects.

The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skin's surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skin's lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skin's low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barrier's robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.

The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters.

Cell adhesion molecules (CAMs) sense the extracellular microenvironment and transmit signals to the intracellular compartment. In this investigation, we addressed the mechanism of signal generation by ectodomains of single-pass transmembrane homophilic CAMs. We analyzed the structure and homophilic interactions of carcinoembryonic antigen (CEA)-related CAM 1 (CEACAM1), which regulates cell proliferation, apoptosis, motility, morphogenesis, and microbial responses. Soluble and membrane-attached CEACAM1 ectodomains were investigated by surface plasmon resonance-based biosensor analysis, molecular electron tomography, and chemical cross-linking. The CEACAM1 ectodomain, which is composed of four glycosylated immunoglobulin-like (Ig) domains, is highly flexible and participates in both antiparallel (trans) and parallel (cis) homophilic binding. Membrane-attached CEACAM1 ectodomains form microclusters in which all four Ig domains participate. Trans-binding between the N-terminal Ig domains increases formation of CEACAM1 cis-dimers and changes CEACAM1 interactions within the microclusters. These data suggest that CEACAM1 transmembrane signaling is initiated by adhesion-regulated changes of cis-interactions that are transmitted to the inner phase of the plasma membrane.

Structural features of the tmRNA-ribosome interaction.

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

Three-dimensional structure of outer hair cell pillars.

UNLABELLED: CONCLUSIONS. Electron tomography was used to generate three-dimensional reconstructions of the pillars that connect the cell membrane with the cytoskeleton of the outer hair cell. Results are consistent with the hypothesis that pillars are important for mechanically linking the membrane with the cytoskeleton. OBJECTIVE: To make a qualitative assessment of the morphology of the sub-membrane pillars of cochlear outer hair cells. MATERIALS AND METHODS: Guinea pig cochleae were fixed and prepared for electron microscopy using protocols described previously. Sections were imaged on an electron microscope equipped with a goniometer. The specimens were tilted through a range of 120°, and an image was acquired at each tilt angle. Filtered back-projection was used to generate three-dimensional reconstructions. RESULTS: Twelve individual pillars were successfully reconstructed. Pillars often connect to the cell membrane through a thin segment, and to the cytoskeleton through a forking structure that may form a central cavity.

Structural basis of hepatocyte growth factor/scatter factor and MET signalling.

The polypeptide growth factor, hepatocyte growth factor/scatter factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade beta-propeller domain of MET harboring the loops connecting the beta-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite "b" face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.

Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography.

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

Morphological variation of individual Escherichia coli 50S ribosomal subunits in situ, as revealed by cryo-electron tomography.

Electron tomography (ET) has been used to reconstruct in situ individual 50S ribosomal subunits in Escherichia coli rifampicin-treated cells. Rifampicin inhibits transcription initiation. As a result, rapid degradation of preformed mRNA and dissociation of 70S ribosomes give accumulation of free subunits. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate the flexible nature of the pathway inside the large ribosomal subunit. In addition, gross morphological heterogeneity was observed in the reconstructions. Our results demonstrate a considerable structural variability among individual 50S subunits in the intracellular environment.

Morphological variation of individual Escherichia coli 30S ribosomal subunits in vitro and in situ, as revealed by cryo-electron tomography.

Cryo-electron tomography has been used to reconstruct the structures of individual ribosomal 30S subunits in Escherichia coli cells treated with rifampicin. Rifampicin inhibits transcription initiation, thus giving depletion of mRNA and accumulation of free 30S and 50S subunits in the cell. Here, we present the 3D morphologies of reconstructed individual 30S ribosomal subunits both in vitro and in situ from E. coli. The head, the platform, and the body of the structures show large conformational movements relative to each other. The particles were grouped into three conformational groups according to the ratio between width and height in the subunit solvent side view. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. The results demonstrate a considerable morphological heterogeneity and structural variability among 30S ribosomal subunits.

Structure and flexibility of individual immunoglobulin G molecules in solution.

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa. Tomograms reveal y-shaped IgG molecules with three protruding subunits. Docking X-ray structures enabled us to recognize the three subunits as two ellipsoidal Fab arms and a heart-shaped Fc stem. Each subunit has a similar structure in the tomograms and in the X-ray map. Notably, the positions of the Fab arms relative to the Fc stem differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. This distribution of individual structures provides a qualitative insight into the system dynamics.

Three-dimensional imaging of in situ specimens with low-dose electron tomography to analyze protein conformation.

We describe a novel three-dimensional (3-D) imaging tool for analysis of protein conformation of in situ samples. Sidec (Sidec Technologies AB, Stockholm, Sweden) electron tomography (SET) uses low-dose electron tomography and a refinement algorithm to reconstruct individual proteins and macromolecular complexes. The approach has successfully reconstructed therapeutic proteins in solution. In this study, we investigate the use of SET to visualize ion channels in cells and tissue samples. SET successfully resolved the volume and structural features of the target complex, showing that it was a tetrameric channel with a central pore. The technology could distinguish and provide 3-D images of the intra- and extracellular domains in the ion channel. In addition, SET was able to show that the channel associates in the form of a tetramer with the four subunits preorganized into dimers. While additional studies using smaller antibody markers are needed to resolve the subunit assembly further, this study demonstrates that SET is a valuable tool for visualization of in situ specimens and can provide important information on the subunit assembly of these macromolecular complexes, and thereby aid in the screening assay process in drug development.

Nephrin promotes cell-cell adhesion through homophilic interactions.

Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+)concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction.