Jens Carlsson

Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase

Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ∼5,000. Here, we show that proton uptake at full speed (∼104 s−1) can be restored in the Asp-132–Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139–Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn2+ addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139–Thr/Asp-132–Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channels

GABAA receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABAA receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABAA receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABAA receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABAA, and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor’s conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABAA receptor ligands.

Proton Uptake and pKa Changes in the Uncoupled Asn139Cys Variant of Cytochrome c Oxidase

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O(2) to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located ~10 Å from the protein surface, is particularly sensitive to mutations that uncouple the O(2) reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They can exist and act as dimers, but the requirement of dimers for agonist-induced signal initiation and structural dynamics remains largely unknown. Frizzled 6 (FZD6) is a member of Class F GPCRs, which bind WNT proteins to initiate signaling. Here, we show that FZD6 dimerizes and that the dimer interface of FZD6 is formed by the transmembrane α-helices four and five. Most importantly, we present the agonist-induced dissociation/re-association of a GPCR dimer through the use of live cell imaging techniques. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of FZD6 signaling to extracellular signal-regulated kinases1/2. The discovery of agonist-dependent dynamics of dimers as an intrinsic process of receptor activation extends our understanding of Class F and other dimerizing GPCRs, offering novel targets for dimer-interfering small molecules.Frizzled 6 (FZD6) is a G protein-coupled receptor (GPCR) involved in several cellular processes. Here, the authors use live cell imaging and spectroscopy to show that FZD6 forms dimers, whose association is regulated by WNT proteins and that dimer dissociation is crucial for FZD6 signaling.

Discovery of trace amine-associated receptor 1 ligands by molecular docking screening against a homology model

Trace Amines (TA) are side-products of the synthesis of classical neurotransmitters within the brain. TAs exert their effect by binding to a family of G protein-coupled receptors termed Trace Amine-Associated Receptors (TAARs). TAAR1 is the best characterised member of this family and studies on TAAR1 have shown that this receptor is a negative regulator of dopamine transmission. Considering the limited number of pharmacological probes available for TAAR1, we aimed to identify novel ligands of this receptor using structure-based virtual screening. A homology model of TAAR1 was generated and over three million commercially available compounds were screened against the orthosteric site using molecular docking. Among the 42 top-ranked compounds that were tested in functional assays, three partial agonists with EC50 values ranging from 1 to 52 μM were discovered. In addition, four potentially weak antagonists were identified. Ten analogs of the two most potent agonists from the screen were also evaluated and three of these displayed equal or greater activity compared to the parent compound. Several of the discovered ligands represent novel scaffolds and are thus promising starting points for development of new pharmacological tools for studying TAAR1 biology.

The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled

Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (IL1) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both β-catenin-dependent and β-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gα12 and Gα13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.

Structure-Guided Screening for Functionally Selective D2 Dopamine Receptor Ligands from a Virtual Chemical Library

Functionally selective ligands stabilize conformations of G protein-coupled receptors (GPCRs) that induce a preference for signaling via a subset of the intracellular pathways activated by the endogenous agonists. The possibility to fine-tune the functional activity of a receptor provides opportunities to develop drugs that selectively signal via pathways associated with a therapeutic effect and avoid those causing side effects. Animal studies have indicated that ligands displaying functional selectivity at the D2 dopamine receptor (D2R) could be safer and more efficacious drugs against neuropsychiatric diseases. In this work, computational design of functionally selective D2R ligands was explored using structure-based virtual screening. Molecular docking of known functionally selective ligands to a D2R homology model indicated that such compounds were anchored by interactions with the orthosteric site and extended into a common secondary pocket. A tailored virtual library with close to 13 000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D2R model. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. A majority of the ligands had comparable maximum effects in the G protein and β-arrestin recruitment assays, but a subset displayed preference for a single pathway. In particular, compound 4 stimulated β-arrestin recruitment (EC50 = 320 nM, Emax = 16%) but had no detectable G protein signaling. The use of structure-based screening and virtual libraries to discover GPCR ligands with tailored functional properties will be discussed.

Fragment optimization for GPCRs by molecular dynamics free energy calculations : Probing druggable subpockets of the A(2A) adenosine receptor binding site

Fragment-based lead discovery is becoming an increasingly popular strategy for drug discovery. Fragment screening identifies weakly binding compounds that require optimization to become high-affinity leads. As design of leads from fragments is challenging, reliable computational methods to guide optimization would be invaluable. We evaluated using molecular dynamics simulations and the free energy perturbation method (MD/FEP) in fragment optimization for the A2A adenosine receptor, a pharmaceutically relevant G protein-coupled receptor. Optimization of fragments exploring two binding site subpockets was probed by calculating relative binding affinities for 23 adenine derivatives, resulting in strong agreement with experimental data (R2 = 0.78). The predictive power of MD/FEP was significantly better than that of an empirical scoring function. We also demonstrated the potential of the MD/FEP to assess multiple binding modes and to tailor the thermodynamic profile of ligands during optimization. Finally, MD/FEP was applied prospectively to optimize three nonpurine fragments, and predictions for 12 compounds were evaluated experimentally. The direction of the change in binding affinity was correctly predicted in a majority of the cases, and agreement with experiment could be improved with rigorous parameter derivation. The results suggest that MD/FEP will become a powerful tool in structure-driven optimization of fragments to lead candidates.

Docking Screens for Dual Inhibitors of Disparate Drug Targets for Parkinson’s Disease

Modulation of multiple biological targets with a single drug can lead to synergistic therapeutic effects and has been demonstrated to be essential for efficient treatment of CNS disorders. However, rational design of compounds that interact with several targets is very challenging. Here, we demonstrate that structure-based virtual screening can guide the discovery of multi-target ligands of unrelated proteins relevant for Parkinsons disease. A library with 5.4 million molecules was docked to crystal structures of the A2A adenosine receptor (A2AAR) and monoamine oxidase B (MAO-B). Twenty-four compounds that were among the highest ranked for both binding sites were evaluated experimentally, resulting in the discovery of four dual-target ligands. The most potent compound was an A2AAR antagonist with nanomolar affinity ( Ki = 19 nM) and inhibited MAO-B with an IC50 of 100 nM. Optimization guided by the predicted binding modes led to the identification of a second potent dual-target scaffold. The two discovered scaffolds were shown to counteract 6-hydroxydopamine-induced neurotoxicity in dopaminergic neuronal-like SH-SY5Y cells. Structure-based screening can hence be used to identify ligands with specific polypharmacological profiles, providing new avenues for drug development against complex diseases.

Scavenging of superoxide by a membrane-bound superoxide oxidase

AbstractSuperoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).The membrane-bound enzyme CybB can directly oxidize superoxide to molecular oxygen and transfer the sequestered electrons to ubiquinone in vitro, providing a mechanism for superoxide scavenging distinct from that of superoxide dismutase.