X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin.

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis.

Trypanosoma cruzi epimastigotes uptake nutrients by endocytosis via the cytostome-cytopharynx complex - an anterior opening (cytostome) continuous with a funnel-shaped invagination (cytopharynx) that extends to the posterior of the cell, accompanied by microtubules. During metacyclogenesis - the transformation of epimastigotes into human-infective metacyclic trypomastigotes - the cytostome-cytopharynx complex disappears, as trypomastigotes lose endocytic ability. To date, no studies have examined cytostome-cytopharynx complex disappearance in detail, or determined if endocytic activity persists during metacyclogenesis. Here, we produced 3D reconstructions of metacyclogenesis intermediates (Ia, Ib, Ic) using electron microscopy tomography and focused ion beam-scanning electron microscopy (FIB-SEM), concentrating on the cytostome-cytopharynx complex and adjacent structures, including the preoral ridge (POR). Parasite endocytic potential was examined by incubation of intermediate forms with the endocytic tracer transferrin (Tf)-Au. Ia, Ib and Ic cells were capable of internalizing Tf-Au, and had a shorter cytopharynx than that of epimastigotes, with the cytostome/POR progressively displaced towards the posterior, following the movement of the kinetoplast/flagellar pocket. While some Ic cells had a short cytopharynx with an enlarged proximal end (∼300nm in diameter, larger than that of the cytostome), other Ic cells had no cytopharynx invagination, but retained the cytopharynx microtubules, which were also present in metacyclics. We conclude that cytostome-cytopharynx disappearance and loss of endocytic ability are late events in metacyclogenesis, during which the cytostome is displaced towards the posterior, probably due to a link to the kinetoplast/flagellar pocket. Retention of the cytopharynx microtubules by metacyclics may allow prompt cytostome-cytopharynx reassembly in amastigotes, upon host cell infection.

Human serum transferrin fibrils: nanomineralisation in bacteria and destruction of red blood cells.

Fibrils formed by human serum transferrin [(1-3 µM) apo-Tf, partially iron-saturated (Fe0.6 -Tf) and holo-Tf (Fe2 -Tf) forms], from dilute bicarbonate solutions, were deposited on formvar surfaces and studied by electron microscopy. We observed that possible bacterial contamination appears to give rise to long, pea-pod-like (PPL) structures for Fe2 -Tf, attributable to the formation of polyhydroxybutyrate (PHB) storage granules, under the nutrient-limiting conditions used. These PPL structures contained periodic nanomineralisation sites susceptible to uranyl stain. Extended incubation of transferrin solutions (about four days) gave rise to extensive transferrin fibril structures. Optical microscopy and AFM studies showed that red blood cells (RBCs) readily adhere to these fibrils. Moreover, the fibrils appear to penetrate RBC membranes and to induce rapid cell destruction (within about 5 h). It is speculated that in situations in vivo where transferrin fibrils can form, such interactions might have adverse physiological consequences, and further studies could aid the understanding of related pathological events.

Structural basis for iron piracy by pathogenic Neisseria.

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

The use of markers for correlative light electron microscopy.

Bioimaging: the visualisation, localisation and tracking of movement of specific molecules in cells using microscopy has become an increasing field of interest within life science research. For this, the availability of fluorescent and electron-dense markers for light and electron microscopy, respectively, is an essential tool to attach to the molecules of interest. In recent years, there has been an increasing effort to combine light and electron microscopy in a single experiment. Such correlative light electron microscopy (CLEM) experiments thus rely on using markers that are both fluorescent and electron dense. Unfortunately, there are very few markers that possess both these properties. Markers for light microscopy such as green fluorescent protein are generally not directly visible in the electron microscopy and vice versa for gold particles. Hence, there has been an intensive search for markers that are directly visible both in the light microscope and in the electron microscope. Here we discuss some of the strategies and pitfalls that are associated with the use of CLEM markers, which might serve as a "warning" that new probes should be extensively tested before use. We focus on the use of CLEM markers for the study of intracellular transport and specifically endocytosis.

Evaluation of imaging plates as recording medium for images of negatively stained single particles and electron diffraction patterns of two-dimensional crystals.

We evaluated imaging plates (IPs) and the DITABIS Micron scanner for their use in recording images of negatively stained single-particle specimens and electron diffraction patterns of two-dimensional crystals. We first established the optimal imaging and read-out conditions for images of negatively stained single-particle specimens using the signal-to-noise ratio of the images as the evaluation criterion. We found that images were best recorded on IPs at a magnification of 67,000x, read out with a gain setting of 20,000 and a laser power setting of 30% with subsequent binning over 2 x 2 pixels. Our results show that for images of negatively stained specimens, for which the resolution is limited to approximately 20 A, IPs are a good alternative to EM film. We also compared IPs with a 2K x 2K Gatan charge-coupled device (CCD) camera for their use in recording electron diffraction patterns of sugar-embedded two-dimensional crystals. Diffraction patterns of aquaporin-0 recorded on IPs and with the CCD camera showed reflections beyond 3 A and had similar R(Friedel) as well as R(merge) values. IPs can thus be used to collect diffraction patterns, but CCD cameras are more convenient and remain the best option for recording electron diffraction patterns.

The Affinity Grid: a pre-fabricated EM grid for monolayer purification.

We have recently developed monolayer purification as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single-particle electron microscopy (EM) without biochemical purification. Here, we introduce the Affinity Grid, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from Escherichia coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50 S subunit rplC. Ribosomal complexes with or without associated mRNA could be prepared depending on the way the sample was applied to the Affinity Grid . Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50 S ribosomal subunit as well as the 70 S ribosome and 30 S ribosomal subunit from images of the same sample. We established that Affinity Grids are stable for some time in the presence of glycerol and detergents, which allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single-particle EM specimens of soluble complexes and membrane proteins.

Monolayer purification: a rapid method for isolating protein complexes for single-particle electron microscopy.

Visualizing macromolecular complexes by single-particle electron microscopy (EM) entails stringent biochemical purification, specimen preparation, low-dose imaging, and 3D image reconstruction. Here, we introduce the "monolayer purification" method, which employs nickel-nitrilotriacetic acid (Ni-NTA) functionalized lipids for simultaneously purifying His-tagged complexes directly from cell lysates while producing specimens suitable for single-particle EM. The method was established by using monolayers containing Ni-NTA lipid to specifically adsorb His-tagged transferrin-transferrin receptor (Tf-TfR) complexes from insect and mammalian cell extracts. The specificity and sensitivity of the method could be improved by adding imidazole to the extracts. The monolayer-purified Tf-TfR samples could be vitrified and used to calculate a 3D reconstruction of the complex. Monolayer purification was then used to rapidly isolate ribosomal complexes from bacteria by overexpressing a single His-tagged ribosomal subunit. The resulting monolayer samples allowed calculation of a cryo-EM 3D reconstruction of the Escherichia coli 50S ribosomal subunit.

Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrin-mediated endocytosis.

Entry of dengue virus 2 (DENV-2) into Aedes albopictus mosquito C6/36 cells was analysed using biochemical and molecular inhibitors, together with confocal and electron microscopy observations. Treatment with monodansylcadaverine, chlorpromazine, sucrose and ammonium chloride inhibited DENV-2 virus yield and protein expression, whereas nystatin, a blocker of caveolae-mediated endocytosis, did not have any effect. Using confocal microscopy, co-localization of DENV-2 E glycoprotein and the marker protein transferrin was observed at the periphery of the cytoplasm. To support the requirement of clathrin function for DENV-2 entry, overexpression of a dominant-negative mutant of Eps15 in C6/36 cells was shown to impair virus entry. The disruption of actin microfilaments by cytochalasin D also significantly affected DENV-2 replication. In contrast, microtubule disruption by colchicine treatment did not impair DENV-2 infectivity, suggesting that DENV-2 does not require transport from early to late endosomes for successful infection of mosquito cells. Furthermore, using transmission electron microscopy, DENV-2 particles of approximately 44-52 nm were found attached within electron-dense invaginations of the plasma membrane and in coated vesicles that resembled those of clathrin-coated pits and vesicles, respectively. Together, these results demonstrate for the first time that DENV-2 enters insect cells by receptor-mediated, clathrin-dependent endocytosis, requiring traffic through an acidic pH compartment for subsequent uncoating and completion of a productive infection.

Exploring transferrin-receptor interactions at the single-molecule level.

Interaction between the iron transporter protein transferrin (Tf) and its receptor at the cell surface is fundamental for most living organisms. Tf receptor (TfR) binds iron-loaded Tf (holo-Tf) and transports it to endosomes, where acidic pH favors iron release. Iron-free Tf (apo-Tf) is then brought back to the cell surface and dissociates from TfR. Here we investigated the Tf-TfR interaction at the single-molecule level under different conditions encountered during the Tf cycle. An atomic force microscope tip functionalized with holo-Tf or apo-Tf was used to probe TfR. We tested both purified TfR anchored to a mica substrate and in situ TfR at the surface of living cells. Dynamic force measurements showed similar results for TfR on mica or at the cell surface but revealed striking differences between holo-Tf-TfR and apo-Tf-TfR interactions. First, the forces necessary to unbind holo-Tf and TfR are always stronger compared to the apo-Tf-TfR interaction. Second, dissociation of holo-Tf-TfR complex involves overcoming two energy barriers, whereas the apo-Tf-TfR unbinding pathway comprises only one energy barrier. These results agree with a model that proposes differences in the contact points between holo-Tf-TfR and apo-Tf-TfR interactions.

omega-Hydrazino linear polyethylenimine: a monoconjugation building block for nucleic acid delivery.

Nonviral gene therapy requires efficient vectors that are able to deliver nucleic acids inside the targeted cell nucleus. Developing new tools for the synthesis of supramolecular vectors with improved transfection efficiency and better biodistribution is therefore a crucial issue. Here we describe the synthesis of a 140-mer linear polyethylenimine (L-PEI) terminated at one end by a highly nucleophilic hydrazine residue. This cationic polymer, whose backbone is well known for its remarkable gene-delivery efficiency, constitutes a building block for omega-regioselective conjugation to molecules through the formation of stable linkages such as the hydrazone bonds. To demonstrate the potential of the omega-hydrazino linear polyethylenimine, human serum transferrin, a ligand that is well know to improve gene-delivery systems, was used as a model of sensitive material. The blood protein was oxidized to generate an aldehyde function and was subsequently conjugated to hydrazino PEI. The new polyethylenimine-transferrin (PEI-Tf) vector was purified and was shown to condense plasmid DNA into compact superstructures compatible with cellular uptake. Finally, the cellular-binding and gene-delivery properties of PEI/DNA polyplexes incorporating different quantities of transferrin were evaluated by FACS analysis and luciferase assay.

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques.

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.

Single particle reconstruction of the human apo-transferrin-transferrin receptor complex.

Most organisms depend on iron as a co-factor for proteins catalyzing redox reactions. Iron is, however, a difficult element for cells to deal with, as it is insoluble in its ferric (Fe3+) form and potentially toxic in its ferrous (Fe2+) form. Thus, in vertebrates iron is transported through the circulation bound to transferrin (Tf) and delivered to cells through an endocytotic cycle involving the transferrin receptor (TfR). We have previously presented a model for the Tf-TfR complex in its iron-bearing form, the diferric transferrin (dTf)-TfR complex [Cheng, Y., Zak, O., Aisen, P., Harrison, S.C., Walz, T., 2004. Structure of the human transferrin receptor-transferrin complex. Cell 116, 565-576]. We have now calculated a single particle reconstruction for the complex in its iron-free form, the apo-transferrin (apoTf)-TfR complex. The same density map was obtained by aligning raw particle images or class averages of the vitrified apoTf-TfR complex to reference models derived from the structures of the dTf-TfR or apoTf-TfR complex. We were unable to improve the resolution of the apoTf-TfR density map beyond 16A, most likely because of significant structural variability of Tf in its iron-free state. The density map does, however, support the model for the apoTf-TfR we previously proposed based on the dTf-TfR complex structure, and it suggests that receptor-bound apoTf prefers to adopt an open conformation.

Nanostructures and molecular force bases of a highly sensitive capacitive immunosensor.

While biosensors have been constructed using various strategies, there is no report describing nanostructures of antibody-immobilized electrode interface in an immunosensor. Here, atomic force microscopy (AFM) and electrochemistry analyses were employed to construct and characterize the nanostructures and electrochemistry of biosensing surface that was created by a sequential self-assembling of bioactive aminobenzenthiol oligomer (o-ABT), glutareldehyde and anti-transferrin (anti-Tf) antibody on the electrode gold surface. Under AFM, a complete coverage of bioactive o-ABT interface could be achieved by anti-Tf antibody at an optimal concentration. The anti-Tf antibody immobilized on electrode surface of the immunosensor exhibited globular-shape topography with some degree of aggregation. Extensive force-curve analysis allowed mapping the functional spots of the anti-Tf immunosensor. Surprisingly, although immunosensing surface was fully covered by anti-Tf antibodies at the optimal concentration, only about 52% of coated anti-Tf antibody molecules (spots) on the electrode surface were able to specifically capture or bind Tf antigen under AFM. Despite limited functional spots, however, the anti-Tf immunosensor was highly specific and sensitive for sensitizing Tf antigen in solution. The anti-Tf molecules on the immunosensor exhibited a greater molecular force bound to holo-Tf (iron-containing form of Tf) than that to apo-Tf (iron-absent form of Tf). Consistently, the anti-Tf immunosensor had a greater electrochemical capacity to sensitize apo-Tf than holo-Tf, supporting the molecular force-based finding by AFM. Thus, the present study elucidated the nanostructures and molecular force bases for the immunosensing capacity of a highly sensitive capacitive immunosensor.

Enhanced antiproliferative activity of transferrin-conjugated paclitaxel-loaded nanoparticles is mediated via sustained intracellular drug retention.

We studied the molecular mechanism of greater efficacy of paclitaxel-loaded nanoparticles (Tx-NPs) following conjugation to transferrin (Tf) ligand in breast cancer cell line. NPs were formulated using biodegradable polymer, poly(lactic-co-glycolide) (PLGA), with encapsulated Tx and conjugated to Tf ligand via an epoxy linker. Tf-conjugated NPs demonstrated greater and sustained antiproliferative activity of the drug in dose- and time-dependent studies compared to that with drug in solution or unconjugated NPs in MCF-7 and MCF-7/Adr cells. The mechanism of greater antiproliferative activity of the drug with conjugated NPs was determined to be due to their greater cellular uptake and reduced exocytosis compared to that of unconjugated NPs, thus leading to higher and sustained intracellular drug levels. The increase in antiproliferative activity of the drug with incubation time in MCF-7/Adr cells with Tf-conjugated NPs suggests that the drug resistance can be overcome by sustaining intracellular drug retention. The intracellular disposition characteristics of Tf-conjugated NPs following their cellular uptake via Tf receptors could have been different from that of unconjugated NPs via nonspecific endocytic pathway, thus influencing the NP uptake, their intracellular retention, and hence the therapeutic efficacy of the encapsulated drug.

Transferrin-mediated gold nanoparticle cellular uptake.

Targeted drug delivery is an important research area in specific therapy. Transferrin-conjugated nanoparticles are an attractive formulation as a vehicle for specific cellular uptake and targeted drug delivery. In this report, atomic force microscopy imaging was used to visualize the process of cellular uptake of transferrin-coupled gold nanoparticles on the surfaces of live cells for the first time. High-resolution images were captured, showing the endocytosis of transferrin-conjugated nanoparticles taking place during the process of internalization. This specific transferrin-mediated nanoparticle uptake was validated by confocal scanning imaging and transferrin competition experiments.

Purification and preliminary X-ray studies on hen serotransferrin in apo- and holo-forms.

Serum transferrins are monomeric glycoproteins with a molecular mass of around 80 kDa, that transport iron to cells via receptor-mediated endocytosis. Although both serum transferrins (STfs) and ovotransferrins (OTfs) are derived from the same gene in aves, the ovotransferrins do not transport iron in vivo. Crystal structures of OTf have been solved, in contrast no three-dimensional structure of avian STf have been determined as yet. Here we report the purification, crystallization, and preliminary crystallographic studies of the hen STf both in apo- (iron free) and holo- (iron loaded) forms. The hen STf has been purified to homogeneity by hydrophobic interaction chromatography. Both the apo- and holo-forms were crystallized by hanging drop vapor diffusion method at 277 K. The apo-crystals diffract to a resolution of 3.0 A and belong to the space group P4(3)2(1)2 with unit cell parameters a=b=90.5 and c=177.9 A. The holo-crystals diffract to a resolution of 2.8 A and belong to space group P2(1) with a=72.8, b=59.6, c=88.2 A, and beta=95.7 degrees.

pH-induced structural changes in human serum apotransferrin. pKa values of histidine residues and N-terminal amino group determined by 1H-NMR spectroscopy.

The binding of apotransferrin (80 kDa) to the transferrin receptor is known to be highly pH-dependent. We have investigated pH-induced structural changes in human serum apotransferrin over the pH* (meter reading in D2O solutions) range 2.5-11 using 1H-NMR spectroscopy. The pKa values of 14 (possibly 15) of the 19 His residues in the protein have been determined as well as that of the terminal amino group (Val1, 7.75). About eight His residues deprotonate when the pH* is raised from the endosomal value of about 5.5 to the blood plasma value (7.4). Four His residues have pKa < 6. Sharp discontinuities in the His titration curves were observed below pH 4.3 and at pH 3.5 molten globule states were detected.