Low-affinity Binding Sites Research Articles

A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival

Apart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue-protective cytokine. In the brain, EPO and its receptor are up-regulated in response to insult and exert pro-survival effects. EPO binds to its receptor (EPOR) via high- and low-affinity binding sites (Sites 1 and 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. Based on the crystal structure of the EPO:EPOR(2) complex, we designed a peptide, termed Epobis, whose sequence encompassed amino acids from binding Site 1. The present study shows that the Epobis peptide specifically binds to EPOR and induces neurite outgrowth from primary neurons in an EPOR-expression dependent manner. Furthermore, Epobis promoted the survival of hippocampal and cerebellar neuronal cultures after kainate treatment and KCl deprivation, respectively. Thus, we identified a new functional agonist of EPOR with the potential to promote neuroregeneration and neuroprotection.

Interaction between Two Sulfate-Conjugated Uremic Toxins,p-Cresyl Sulfate and Indoxyl Sulfate, during Binding with Human Serum Albumin

Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 10(5) M(-1) (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.

Transient CPEB dimerization and translational control

During oocyte development, the cytoplasmic polyadenylation element-binding protein (CPEB) nucleates a set of factors on mRNA that controls cytoplasmic polyadenylation and translation. The regulation of polyadenylation is mediated in part through serial phosphorylations of CPEB, which control both the dynamic integrity of the cytoplasmic polyadenylation apparatus and CPEB stability, events necessary for meiotic progression. Because the precise stoichiometry between CPEB and CPE-containing RNA is responsible for the temporal order of mRNA polyadenylation during meiosis, we hypothesized that, if CPEB production exceeded the amount required to bind mRNA, the excess would be sequestered in an inactive form. One attractive possibility for the sequestration is protein dimerization. We demonstrate that not only does CPEB form a dimer, but dimerization requires its RNA-binding domains. Dimer formation prevents CPEB from being UV cross-linked to RNA, which establishes a second pool of CPEB that is inert for polyadenylation and translational control. During oocyte maturation, the dimers are degraded much more rapidly than the CPEB monomers, due to their greater affinity for polo-like kinase 1 (plx1) and the ubiquitin E3 ligase β-TrCP. Because dimeric CPEB also binds cytoplasmic polyadenylation factors with greater affinity than monomeric CPEB, it may act as a hub or reservoir for the polyadenylation machinery. We propose that the balance between CPEB and its target mRNAs is maintained by CPEB dimerization, which inactivates spare proteins and prevents them from inducing polyadenylation of RNAs with low affinity binding sites. In addition, the dimers might serve as molecular hubs that release polyadenylation factors for translational activation upon CPEB dimer destruction.

Comparison of solid phase microextraction versus spectroscopic techniques for binding studies of carbamazepine

The binding of carbamazepine to human serum albumin was studied in vitro using solid-phase microextraction (SPME) with liquid chromatography-ultraviolet detection (LC-UV), as well as spectroscopic fluorescence and nuclear magnetic resonance ((1)H NMR) techniques. We were able to recognize one high affinity binding site with both fluorescence and SPME methods. Additionally, SPME experiment showed the existence of one lower affinity binding site for carbamazepine at the range of concentrations studied with fluorescence. The analysis of Hill's plot indicated positive cooperativity between drugs located in these two binding sites. Two low affinity-binding sites have been found with SPME-LC-UV analysis performed in parallel to (1)H NMR study, which does not show any complex formation. In conclusion, the results of the studies with carbamazepine as a model drug showed the advantages of simultaneous use of solid phase microextraction and spectroscopic methods in protein binding studies and indicated complementary information, which can be obtained with the use of SPME. Furthermore, we show that SPME in combination with liquid chromatography-mass spectrometry permitted direct in vitro determination of plasma-protein binding and direct in vivo evaluation of inter-animal variability in free concentrations of carbamazepine at physiologically relevant concentrations, the type of experiments typically inaccessible by spectroscopic techniques due to poor sensitivity and different mode of implementation.

The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA

The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65(CTD) is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65(CTD) interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem-loop IV.

Structural Analysis and Stochastic Modelling Suggest a Mechanism for Calmodulin Trapping by CaMKII

Activation of CaMKII by calmodulin and the subsequent maintenance of constitutive activity through autophosphorylation at threonine residue 286 (Thr286) are thought to play a major role in synaptic plasticity. One of the effects of autophosphorylation at Thr286 is to increase the apparent affinity of CaMKII for calmodulin, a phenomenon known as “calmodulin trapping”. It has previously been suggested that two binding sites for calmodulin exist on CaMKII, with high and low affinities, respectively. We built structural models of calmodulin bound to both of these sites. Molecular dynamics simulation showed that while binding of calmodulin to the supposed low-affinity binding site on CaMKII is compatible with closing (and hence, inactivation) of the kinase, and could even favour it, binding to the high-affinity site is not. Stochastic simulations of a biochemical model showed that the existence of two such binding sites, one of them accessible only in the active, open conformation, would be sufficient to explain calmodulin trapping by CaMKII. We can explain the effect of CaMKII autophosphorylation at Thr286 on calmodulin trapping: It stabilises the active state and therefore makes the high-affinity binding site accessible. Crucially, a model with only one binding site where calmodulin binding and CaMKII inactivation are strictly mutually exclusive cannot reproduce calmodulin trapping. One of the predictions of our study is that calmodulin binding in itself is not sufficient for CaMKII activation, although high-affinity binding of calmodulin is.

Hemolymph ion regulation and kinetic characteristics of the gill (Na+, K+)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na(+), K(+))-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg(-1) H(2)O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg(-1) H(2)O). Hemolymph [Na(+)] (323.0 ± 2.5 mmol L(-1)) and [Mg(2+)] (34.6 ± 1.0 mmol L(-1)) are hypo-regulated while [Ca(2+)] (22.5 ± 0.7 mmol L(-1)) is hyper-regulated; [K(+)] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L(-1)) but hypo-regulated (6.2 ± 0.7 mmol L(-1)) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm=46.5 ± 3.5 Umg(-1); K(0.5)=7.07 ± 0.01 μmol L(-1)) and a low-affinity ATP binding site (Vm=108.1 ± 2.5 U mg(-1); K(0.5)=0.11 ± 0.3 mmol L(-1)), both obeying cooperative kinetics, were disclosed. Modulation of (Na(+), K(+))-ATPase activity by Mg(2+), K(+) and NH(4)(+) also exhibits site-site interactions, but modulation by Na(+) shows Michaelis-Menten kinetics. (Na(+), K(+))-ATPase activity is synergistically stimulated up to 45% by NH(4)(+) plus K(+). Enzyme catalytic efficiency for variable [K(+)] and fixed [NH(4)(+)] is 10-fold greater than for variable [NH(4)(+)] and fixed [K(+)]. Ouabain inhibited ≈80% of total ATPase activity (K(I)=464.7 ± 23.2 μmol L(-1)), suggesting that ATPases other than (Na(+), K(+))-ATPase are present. While (Na(+), K(+))-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1)mg(-1)) and 45‰-acclimated crabs (around 154 nmol Pi min(-1)mg(-1)), ATP affinity decreases 110-fold and Na(+) and K(+) affinities increase 2-3-fold in 45‰-acclimated crabs.

Single-Molecule Fluorescence Spectroscopy of the Structure and Dynamics of the Spliceosomal Complex

Binding reactions can be ideally investigated by Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS). Here, this method is used to study the recruitment and dissociation of proteins to and from the spliceosome. The spliceosome is the cellular machinery responsible for removing non-coding introns from precursor mRNA (pre-mRNA). The spliceosome assembles on a pre-mRNA, which consists of two exons and one intron, stepwise by the binding of five snRNPs and several other proteins. This leads to the formation of the B complex which has no active catalytic center yet. Structural rearrangements lead to the activated Bact complex which is formed by further rearrangements in the catalytically active B* complex. The B* complex catalyzes the first splicing step in which the branch site (BS) attacks the 5' splice site (5'SS). The 5' exon is cleaved and the intron-3'-exon lariat is formed. The resulting C complex then catalyzes the second splicing step. The intron is cleaved and both exons are joined together forming mature mRNA. During both catalytic steps, the spliceosome undergoes many changes in its protein composition, and several structural rearrangements occur. These changes and rearrangements can be investigated by standard biochemical methods. However, these methods often do not provide data about dynamics, which are necessary for understanding the recruitment and release of particular molecules. One method which provides these information is FCCS. In contrast to methods like mass spectrometry, in FCCS measurements it is possible to observe reactions in real-time and at equilibrium without further biochemical perturbation of the sample. FCCS works at low nanomolar concentrations and only small sample volumes are necessary. Using FCCS, it could be determined how changes in the spliceosome composition and conformation occur (simultaneously or consecutively). The roles of certain spliceosomal RNA helicases in the restructuring of the complex could be investigated. FCCS enables the observation of protein-protein interactions and the determination of binding constants for proteins to the spliceosome. In order to better understand the dynamic nature of the spliceosome during its catalytic activation, the step 1 factor Cwc25 and the step 2 factors Slu7, Prp18, and Prp16 were investigated. Their role in the maturation process, the fundamental question of the time point and manner of their recruitment has not been answered yet. With FCCS, the binding of the step 1 factor Cwc25 was observed. It was shown that Cwc25 has a high-binding affinity to the spliceosome after the Prp2-mediated rearrangements into the catalytically activated B* complex. The high-binding affinity was reflected in a strong binding constant of 30 pM which was measured with FCCS. By using several mutant pre-mRNAs it could be demonstrated that Cwc25's release does not depend on the second catalytic step per se. Its dissociation depends on the docking of the 3'SS to the active site and the action of Prp16 and Slu7/Prp18. It was also observed that the distance between the branch site and 3'SS influences the release of Cwc25 and the second catalytic step. If the distance between BS and 3'SS is short (e.g. 7 nucleotides long), the distance between the 3'SS and the active site is also small. The 3'SS can dock into the active site without the further stabilization by Slu7/Prp18. The activity of Prp16 and the docking event lead to the release of Cwc25. Slu7/Prp18 can stabilize the system and thereby induce further release of Cwc25. If the distance between BS and 3'SS is longer (e.g. 38 nucleotides), Slu7/Prp18 has to stabilize the interaction of the 3'SS and the active site and the step 2 conformation. In this case, Prp16 alone cannot induce the release of Cwc25. Cwc25 does dissociate from the spliceosome after the action of Prp16 and in the presence of Slu7/Prp18. It was shown further that Prp16 and its ATPase activity are necessary for the formation of a functional step 2 active site. Slu7/Prp18 are required for the efficient docking of the 3' splice site (3'SS) to the active site. The FCCS experiments showed that Prp16 and Slu7/Prp18 have distinct binding sites in the spliceosome which are formed during the catalytic activation. During activation their first low-affinity binding sites are transformed into high-affinity binding sites. Both proteins are bound to the spliceosome at an early stage so that they are present before their catalytic function is actually required in the process. Here, it was shown that FCCS is ideally suited to investigate macromolecular protein complexes like the spliceosome and is a powerful tool for studying quantitatively spliceosomal protein dynamics at equilibrium.

A New View on Allosteric Modulation of Alpha 7 Nicotinic Acetylcholin Receptors

Nicotinic acetylcholine receptors (nAChR) are ligand gated cation channels that are opened by agonists like Acetylcholine and Nicotine. They are widely distributed in neurons all over central nervous system, where they are mainly found at postsynaptic locations of cholinergic nerve endings.The most abundant homomeric nicotinic acetylcholine receptors in the mammalian brain are the pentameric alpha7 nAChRs which consist of five alpha7 subunits. Each subunit provides an orthosteric low affinity binding site for its endogenous ligand, acetylcholine. Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. Alpha 7 receptors are also a target for non-CNS indications like diabetes and asthma.View Large Image | View Hi-Res Image | Download PowerPoint SlideHere we show the results of the screen of allosteric modulators on alpha 7 receptors. High parallelization without a compromise in data quality combined with fast and accurate solution exchange allows characterization of many compounds. A second analysis at physiological temperatures showed a pronounced effect on the allosteric modulation.

A model of spatially restricted transcription in opposing gradients of activators and repressors.

Morphogens control patterns of transcription in development, often by establishing concentration gradients of a single transcriptional activator. However, many morphogens, including Hedgehog, create opposing activator and repressor gradients (OARGs). In contrast to single activator gradients, it is not well understood how OARGs control transcriptional patterns. We present a general thermodynamic model that explains how spatial patterns of gene expression are established within OARGs. The model predicts that differences in enhancer binding site affinities for morphogen-responsive transcription factors (TFs) produce discrete transcriptional boundaries, but only when either activators or repressors bind cooperatively. This model quantitatively predicts the boundaries of gene expression within OARGs. When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila. Because our model is general, it may explain the role of low-affinity binding sites in many contexts, including mammalian Hedgehog gradients.

Energy-Independent Helicase Activity of a Viral Genome Packaging Motor

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.

Binding of kebuzone to human serum albumin studied by high performance liquid affinity chromatography.

The binding of the anti-inflammatory agent kebuzone (CAS 853-34-9) to human serum albumin (HSA) was examined by means of high performance liquid affinity chromatography. Phenylbutazone and diazepam, the binding properties of which were an object of our previous work, were used as markers for the major supposed binding sites on HSA. It was established that kebuzone binds to the high-affinity phenylbutazone binding sites as well as to the low-affinity diazepam binding sites. The estimated values of the equilibrium affinity constants were 5.9 x 10(4) l/mol and 1.7 x 10(3) l/mol, respectively, suggesting a low binding affinity for kebuzone. A stepwise mechanism of the binding process could be proposed: primary at phenylbutazone sites, and secondary at the diazepam sites. The binding behavior of kebuzone was very similar to that of the structurally related compound phenylbutazone. The observed lower affinity is well explained in view of the lower lipophilicity of kebuzone.

Coordination chemistry of copper proteins: How nature handles a toxic cargo for essential function

Biological copper is coordinated predominantly by just three ligand types: the side chains of histidine, cysteine, and methionine, with of course some exceptions. The arrangement of these components, however, is fascinating. The diversity provided by just these three ligands provides choices of nitrogen vs. sulfur, neutral vs. charged, hydrophilic vs. hydrophobic, susceptibility to oxidation, and degree of pH-sensitivity. In this review we examine how the total number of ligands, their spatial arrangement and solvent accessibility, the various combinations of imidazole, thiolate, and thioether donors, all work together to provide binding sites that either enable copper to carry out a function, or safely transport it in a way that prevents toxic reactivity. We separate copper proteins into two broad classes, those that utilize the metal as a cofactor, or those that traffic the metal. Enzymes and proteins that utilize copper as a cofactor use high affinity sites of high coordination numbers of 4–5 that prevent loss of the metal during redox cycling. Copper trafficking proteins, on the other hand, promote metal transfer either by having low affinity binding sites with moderate coordination number ~ 4, or by having lower coordinate binding sites of 2–3 ligands that bind with high affinity. Both strategies retain the metal but allow transfer under appropriate conditions. Analysis of studies from our own lab on model peptides, combined with those from other labs, raises an interesting hypothesis that various methionine/histidine/cysteine combinations provide organisms with dynamic, multifunctional domains on copper trafficking proteins that facilitate copper transfer under different extracellular, subcellular, and tissue-specific scenarios of pH, redox environment, and presence of other copper carriers or target proteins.

SecA Alone Can Promote Protein Translocation and Ion Channel Activity: SecYEG INCREASES EFFICIENCY AND SIGNAL PEPTIDE SPECIFICITY

SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1-2 μM. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity.

Characterization of COR627 and COR628, Two Novel Positive Allosteric Modulators of the GABAB Receptor

The potential efficacy of GABA(B) receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA(B) receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA(B) receptor may constitute a novel approach in the pharmacological manipulation of the GABA(B) receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA(B) PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding to native GABA(B) receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA(B) receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([(3)H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA(B) receptor.

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

A Two-in-One Antibody against HER3 and EGFR Has Superior Inhibitory Activity Compared with Monospecific Antibodies

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling invitro and invivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.

New insights into tau–microtubules interaction revealed by isothermal titration calorimetry

Microtubule dynamic instability is tightly regulated by coordinated action of stabilizing and destabilizing microtubule associated proteins. Among the stabilizing proteins, tau plays a pivotal role in both physiological and pathological processes. Nevertheless, the detailed mechanism of tau–tubulin interaction is still subject to controversy. In this report, we studied for the first time tau binding to tubulin by a direct thermodynamic method in the absence of any tubulin polymerization cofactors that could influence this process. Isothermal titration calorimetry enabled us to evidence two types of tau–tubulin binding modes: one corresponding to a high affinity binding site with a tau:tubulin stoichiometry of 0.2 and the other one to a low affinity binding site with a stoichiometry of 0.8. The same stoichiometries were obtained at all temperatures tested (10–37°C), indicating that the mechanism of interaction does not depend on the type of tubulin polymer triggered upon tau binding. These findings allowed us to get new insights into the topology of tau on microtubules.

Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K D of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K D of 0.14 nM and for the high-affinity insulin binding sites with K D of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min −1 and 0.013 min −1, respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells.