Renyu Liu

Structural basis for high-affinity volatile anesthetic binding in a natural 4-helix bundle protein

Physiologic sites for inhaled anesthetics are presumed to be cavities within transmembrane 4‐α‐helix bundles of neurotransmitter receptors, but confirmation of binding and structural detail of such sites remains elusive. To provide such detail, we screened soluble proteins containing this structural motif, and found only one that exhibited evidence of strong anesthetic binding. Ferritin is a 24‐mer of 4‐α‐helix bundles; both halothane and isoflurane bind with KA values of ∼105 M−1, higher than any previously reported inhaled anesthetic‐protein interaction. The crystal structures of the halothane/apoferritin and isoflurane/apoferritin complexes were determined at 1.75 Å resolution, revealing a common anesthetic binding pocket within an interhelical dimerization interface. The high affinity is explained by several weak polar contacts and an optimal host/guest packing relationship. Neither the acidic protons nor ether oxygen of the anesthetics contribute to the binding interaction. Compared with unliganded apoferritin, the anesthetic produced no detectable alteration of structure or B factors. The remarkably high affinity of the anesthetic/apoferritin complex implies greater selectivity of protein sites than previously thought, and suggests that direct protein actions may underlie effects at lower than surgical levels of anesthetic, including loss of awareness.—Liu, R., Loll, P. J. Eckenhoff, R. G. Structural basis for high‐affinity volatile anesthetic binding in a natural 4‐helix bundle protein. FASEB J. 19, 567–576 (2005)

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABAA receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABAA receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

Scalable Production of Highly Sensitive Nanosensors Based on Graphene Functionalized with a Designed G Protein-Coupled Receptor

We have developed a novel, all-electronic biosensor for opioids that consists of an engineered μ-opioid receptor protein, with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor to read out ligand binding. A variant of the receptor protein that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. A shadow mask process was developed to fabricate arrays of hundreds of graphene transistors with average mobility of ∼1500 cm2 V–1 s–1 and yield exceeding 98%. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, an opioid receptor antagonist, with a detection limit of 10 pg/mL.

Comparative binding character of two general anaesthetics for sites on human serum albumin

Propofol and halothane are clinically used general anaesthetics, which are transported primarily by HSA (human serum albumin) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of these drugs. We characterized anaesthetic-HSA interactions in solution using elution chromatography, ITC (isothermal titration calorimetry), hydrogen-exchange experiments and geometric analyses of high-resolution structures. Binding affinity of propofol to HSA was determined to have a K(d) of 65 microM and a stoichiometry of approx. 2, whereas the binding of halothane to HSA showed a K(d) of 1.6 mM and a stoichiometry of approx. 7. Anaesthetic-HSA interactions are exothermic, with propofol having a larger negative enthalpy change relative to halothane. Hydrogen-exchange studies in isolated recombinant domains of HSA showed that propofol-binding sites are primarily found in domain III, whereas halothane sites are more widely distributed. Both location and stoichiometry from these solution studies agree with data derived from X-ray crystal-structure studies, and further analyses of the architecture of sites from these structures suggested that greater hydrophobic contacts, van der Waals interactions and hydrogen-bond formation account for the stronger binding of propofol as compared with the less potent anaesthetic, halothane.

A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor: The Human Mu Opioid Receptor

G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K d  = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.

Novel Molecular Targets of Dezocine and Their Clinical Implications

Background:Although dezocine is a partial &mgr;-opioid receptor agonist, it is not a controlled substance. Thus, the characterization of the molecular targets of dezocine is critical for scientific and clinical implications. The goal of this study is to characterize molecular targets for dezocine and determine their implications. Methods:A binding screen for dezocine was performed on 44 available receptors and transporter proteins. Functional assays for the novel targets were performed along with computation calculations to locate the binding site. A G protein activation study was performed for the human &kgr; opioid receptor to determine whether dezocine is a &kgr;-antagonist. Data are presented as mean ± standard error. Results:The affinities for dezocine were 3.7 ± 0.7 nM for the &mgr; receptor, 527 ± 70 nM for the &dgr;-receptor, and 31.9 ± 1.9 nM for the &kgr;-receptor. Dezocine failed to induce G protein activation with &kgr;-opioid receptor and concentration dependently inhibited &kgr;-agonist (salvinorin A and nalbuphine)–induced receptor activation, indicating that dezocine is a &kgr;-antagonist. Two novel molecular targets (norepinephrine transporter and serotonin transporter) were identified. Dezocine concentration-dependently inhibited norepinephrine and serotonin reuptake in vitro. The half maximal inhibitory concentrations (expressed as pIC50) were 5.68 ± 0.11 for norepinephrine transporter and 5.86 ± 0.17 for serotonin transporter. Dezocine occupied the binding site for known norepinephrine transporter and serotonin transporter inhibitors. Conclusions:The unique molecular pharmacological profile of dezocine as a partial &mgr;-receptor agonist, a &kgr;-receptor antagonist, and a norepinephrine and serotonin reuptake inhibitor (via norepinephrine transporter and serotonin transporter) was revealed. These discoveries reveal potentially important novel clinical implications and drug interactions of dezocine.

Scalable Production of Molybdenum Disulfide Based Biosensors

We demonstrate arrays of opioid biosensors based on chemical vapor deposition grown molybdenum disulfide (MoS2) field effect transistors (FETs) coupled to a computationally redesigned, water-soluble variant of the μ-opioid receptor (MOR). By transferring dense films of monolayer MoS2 crystals onto prefabricated electrode arrays, we obtain high-quality FETs with clean surfaces that allow for reproducible protein attachment. The fabrication yield of MoS2 FETs and biosensors exceeds 95%, with an average mobility of 2.0 cm(2) V(-1) s(-1) (36 cm(2) V(-1) s(-1)) at room temperature under ambient (in vacuo). An atomic length nickel-mediated linker chemistry enables target binding events that occur very close to the MoS2 surface to maximize sensitivity. The biosensor response calibration curve for a synthetic opioid peptide known to bind to the wild-type MOR indicates binding affinity that matches values determined using traditional techniques and a limit of detection ∼3 nM (1.5 ng/mL). The combination of scalable array fabrication and rapid, precise binding readout enabled by the MoS2 transistor offers the prospect of a solid-state drug testing platform for rapid readout of the interactions between novel drugs and their intended protein targets.

Salvinorin A Produces Cerebrovasodilation through Activation of Nitric Oxide Synthase, κ Receptor, and Adenosine Triphosphate–sensitive Potassium Channel

Background:Salvinorin A is a nonopioid, selective &kgr; opioid–receptor agonist. Despite its high potential for clinical application, its pharmacologic profile is not well known. In the current study, we hypothesized that salvinorin A dilates pial arteries via activation of nitric oxide synthase, adenosine triphosphate–sensitive potassium channels, and opioid receptors. Methods:Cerebral artery diameters and cyclic guanosine monophosphate in cortical periarachnoid cerebrospinal fluid were monitored in piglets equipped with closed cranial windows. Observation took place before and after salvinorin A administration in the presence or absence of an opioid antagonist (naloxone), a &kgr; opioid receptor–selective antagonist (norbinaltorphimine), nitric oxide synthase inhibitors (N(G)-nitro-L-arginine and 7-nitroindazole), a dopamine receptor D2 antagonist (sulpiride), and adenosine triphosphate–sensitive potassium and Ca2+-activated K channel antagonists (glibenclamide and iberiotoxin). The effects of salvinorin A on the constricted cerebral artery induced by hypocarbia and endothelin were investigated. Data were analyzed by repeated measures ANOVA (n = 5) with statistical significance set at a P value of less than 0.05. Results:Salvinorin A induced immediate but brief vasodilatation that was sustained for 30 min via continual administration every 2 min. Vasodilatation and the associated cyclic guanosine monophosphate elevation in cerebrospinal fluid were abolished by preadministration N(G)-nitro-L-arginine, but not 7-nitroindazole. Although naloxone, norbinaltorphimine, and glibenclamide abolished salvinorin A–induced cerebrovasodilation, this response was unchanged by iberiotoxin and sulpiride. Hypocarbia and endothelin-constricted pial arteries responded similarly to salvinorin A, to the extent observed under resting tone. Conclusions:Salvinorin A dilates cerebral arteries via activation of nitric oxide synthase, adenosine triphosphate–sensitive potassium channel, and the &kgr; opioid receptor.

Salvinorin A Administration after Global Cerebral Hypoxia/Ischemia Preserves Cerebrovascular Autoregulation via Kappa Opioid Receptor in Piglets

Background Cerebral hypoxia/ischemia (HI) is not uncommon during the perinatal period. If occurring, it can result in severe neurologic disabilities that persist throughout life. Salvinorin A, a non-opioid Kappa opioid receptors (KOR) selective agonist, has the potential to address this devastating situation. We have demonstrated that salvinorin A administration before HI, preserves pial artery autoregulative function through both the KOR and extracellular signal-regulated kinases (ERK) pathways. In the present study, we tested the hypothesis that administration of salvinorin A after HI could preserve cerebral autoregulation via KOR and ERK pathway. Methodology/Principal Findings The response of the pial artery to hypercapnia, hypotension and isoproterenol were monitored before and 1 hour after HI in piglets equipped with a cranial window. Four groups of drug administration were performed after HI. The control group had DMSO (1 µl/kg, i.v.) administrated immediately after HI. Two salvinorin A treated groups had salvinorin A (10 µg/kg, i.v.) administrated 0 and 30 min after HI, respectively. The 4th group had salvinorin A and the KOR antagonist norbinaltorphimine (Nor-BIN, 1 µM topical) co-administrated 0 min after HI (n = 5). The dilation responses of the pial artery to hypercapnia and hypotension were impaired after global HI and were preserved with salvinorin A administration immediately or 30 min after HI. The preservation of autoregulation was abolished when nor-BIN was administered. Levels of phosphor-ERK(pERK)/ERK in the cerebrospinal fluid (CSF) were measured before and 1 hour after HI. After HI, the pERK/ERK levels significantly increased in both DMSO control group and salvinorin A and nor-BIN co-administration group. The elevated levels of pERK/ERK were not observed with salvinorin A only groups. Conclusions Salvinorin A administration 0 and 30 min after HI preserves autoregulation of pial artery to hypercapnia and hypotension via kappa opioid receptor and ERK pathway.

Ferritin couples iron and fatty acid metabolism

A physiological relationship between iron, oxidative injury, and fatty acid metabolism exists, but transduction mechanisms are unclear. We propose that the iron storage protein ferritin contains fatty acid binding sites whose occupancy modulates iron uptake and release. Using isothermal microcalorimetry, we found that arachidonic acid binds ferritin specifically and with 60 μM affinity. Arachidonate binding by ferritin enhanced iron mineralization, decreased iron release, and protected the fatty acid from oxidation. Cocrystals of arachidonic acid and horse spleen apoferritin diffracted to 2.18 Å and revealed specific binding to the 2‐fold intersubunit pocket. This pocket shields most of the fatty acid and its double bonds from solvent but allows the arachidonate tail to project well into the ferrihydrite mineralization site on the ferritin L‐subunit, a structural feature that we implicate in the effects on mineralization by demonstrating that the much shorter saturated fatty acid, caprylate, has no significant effects on mineralization. These combined effects of arachidonate binding by ferritin are expected to lower both intracellular free iron and free arachidonate, thereby providing a previously unrecognized mechanism for limiting lipid peroxidation, free radical damage, and proinflammatory cascades during times of cellular stress.—Bu, W., Liu, R., Cheung‐Lau, J. C., Dmochowski, I. J., Loll, P. J., Eckenhoff, R. G. Ferritin couples iron and fatty acid metabolism. FASEB J. 26, 2394‐2400 (2012). www.fasebj.org