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Purpose: OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter in the liver and a key mediator of xenobiotic clearance. However, the regulatory mechanisms that determine OATP1B1 activity remain uncertain, and as a result, unexpected drug–drug interactions involving OATP1B1 substrates continue to be reported, including several involving tyrosine kinase inhibitors (TKI). Experimental Design: OATP1B1-mediated activity in overexpressing HEK293 cells and hepatocytes was assessed in the presence of FDA-approved TKIs, while rosuvastatin pharmacokinetics in the presence of an OATP1B1 inhibiting TKI were measured in vivo. Tyrosine phosphorylation of OATP1B1 was determined by LC/MS-MS–based proteomics and transport function was measured following exposure to siRNAs targeting 779 different kinases. Results: Twenty-nine of 46 FDA-approved TKIs studied significantly inhibit OATP1B1 function. Inhibition of OATP1B1 by TKIs, such as nilotinib, is predominantly noncompetitive, can increase systemic concentrations of rosuvastatin in vivo, and is associated with reduced phosphorylation of OATP1B1 at tyrosine residue 645. Using genetic screens and functional validation studies, the Src kinase LYN was identified as a potential regulator of OATP1B1 activity that is highly sensitive to inhibition by various TKIs at clinically relevant concentrations. Conclusions: A novel kinase-dependent posttranslational mechanism of OATP1B1 activation was identified and interference with this process by TKIs can influence the elimination of a broad range of xenobiotic substrates.

Significance Site-directed fluorometry was used to understand conformational changes of the Na+/glucose symporter. SGLT1 functions by a mechanism where the substrate-binding site alternates between the two faces of the membrane, but little is known about the underlying conformational changes. Rhodamines were covalently inserted into the substrate cavity, and changes of fluorescence were measured in real time with the opening and closing of the outer gate as SGLT1 was driven between inward and outward conformations using voltage jumps. Structural modeling indicated that the quenching with gating opening was due to an increased solvation of rhodamine and an increase in polar residues lining the wall of the cavity. This experimental approach will lead to a better understanding of the mechanism of membrane transport., In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

International audience; The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

Amalgam (Ama) is a secreted neuronal adhesion protein that contains three tandem immunoglobulin domains. It has both homophilic and heterophilic cell adhesion properties, and is required for axon guidance and fasciculation during early stages of Drosophila development. Here, we report its biophysical characterization and use small-angle x-ray scattering to determine its low-resolution structure in solution. The biophysical studies revealed that Ama forms dimers in solution, and that its secondary and tertiary structures are typical for the immunoglobulin superfamily. Ab initio and rigid-body modeling by small-angle x-ray scattering revealed a distinct V-shaped dimer in which the two monomer chains are aligned parallel to each other, with the dimerization interface being formed by domain 1. These data provide a structural basis for the dual adhesion characteristics of Ama. Thus, the dimeric structure explains its homophilic adhesion properties. Its V shape suggests a mechanism for its interaction with its receptor, the single-pass transmembrane adhesion protein neurotactin, in which each “arm” of Ama binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane.

In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 A resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ∼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and Abeta peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 A resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the "anionic" subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N(epsilon2), which is hydrogen-bonded to Ser200O(gamma) in the native enzyme. The second nor-meptazinol binds at the peripheral "anionic" site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors.

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high beta-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R(H)=16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.