Structural insights into the inhibition of glycine reuptake.

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

Generation of synthetic nanobodies against delicate proteins.

Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.

Biotinylation of Membrane Proteins for Binder Selections.

The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.

Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.

Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development.

Structural Basis for Allosteric Ligand Recognition in the Human CC Chemokine Receptor 7.

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.

Development of an antibody fragment that stabilizes GPCR/G-protein complexes.

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gßγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.

Ligand channel in pharmacologically stabilized rhodopsin.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.

Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein.

The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system.

Structure of the acid-sensing ion channel 1 in complex with the gating modifier Psalmotoxin 1.

Venom-derived peptide toxins can modify the gating characteristics of excitatory channels in neurons. How they bind and interfere with the flow of ions without directly blocking the ion permeation pathway remains elusive. Here we report the crystal structure of the trimeric chicken Acid-sensing ion channel 1 in complex with the highly selective gating modifier Psalmotoxin 1 at 3.0 Å resolution. The structure reveals the molecular interactions of three toxin molecules binding at the proton-sensitive acidic pockets of Acid-sensing ion channel 1 and electron density consistent with a cation trapped in the central vestibule above the ion pathway. A hydrophobic patch and a basic cluster are the key structural elements of Psalmotoxin 1 binding, locking two separate regulatory regions in their relative, desensitized-like arrangement. Our results provide a general concept for gating modifier toxin binding suggesting that both surface motifs are required to modify the gating characteristics of an ion channel.

Structure and mechanism of ABC transporter proteins.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP. The crystal structures of four ABC transporters have recently been determined. They reveal similar arrangements of the conserved ATP-hydrolyzing nucleotide-binding domains, but unrelated architectures of the transmembrane domains, with the notable exception of a common 'coupling helix' that is essential for transmitting conformational changes. The structures suggest a mechanism that rationalizes ATP-driven transport: While binding of ATP appears to trigger an outward-facing conformation, dissociation of the hydrolysis products may promote an inward-facing conformation. This basic scheme can, in principle, explain nutrient import by ABC importers and drug extrusion by ABC exporters.

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism.

ATP-binding cassette (ABC) transporters are integral membrane proteins that move diverse substrates across cellular membranes. ABC importers catalyse the uptake of essential nutrients from the environment, whereas ABC exporters facilitate the extrusion of various compounds, including drugs and antibiotics, from the cytoplasm. How ABC transporters couple ATP hydrolysis to the transport reaction has long remained unclear. The recent crystal structures of four complete ABC transporters suggest that a key step of the molecular mechanism is conserved in importers and exporters. Whereas binding of ATP promotes an outward-facing conformation, the release of the hydrolysis products ADP and phosphate promotes an inward-facing conformation. This basic scheme can in principle explain ATP-driven drug export and binding protein-dependent nutrient uptake.

Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP.

Staphylococcus aureus Sav1866 is a bacterial homolog of the human ABC transporter Mdr1 that causes multidrug resistance in cancer cells. We report the crystal structure of Sav1866 in complex with adenosine-5'-(beta,gamma-imido)triphosphate (AMP-PNP) at 3.4A resolution and compare it with the previously determined structure of Sav1866 with bound ADP. Besides differences in the ATP-binding sites, no significant conformational changes were observed. The results confirm that the ATP-bound state of multidrug ABC transporters is coupled to an outward-facing conformation of the transmembrane domains.

Structure of a bacterial multidrug ABC transporter.

Multidrug transporters of the ABC family facilitate the export of diverse cytotoxic drugs across cell membranes. This is clinically relevant, as tumour cells may become resistant to agents used in chemotherapy. To understand the molecular basis of this process, we have determined the 3.0 A crystal structure of a bacterial ABC transporter (Sav1866) from Staphylococcus aureus. The homodimeric protein consists of 12 transmembrane helices in an arrangement that is consistent with cross-linking studies and electron microscopic imaging of the human multidrug resistance protein MDR1, but critically different from that reported for the bacterial lipid flippase MsbA. The observed, outward-facing conformation reflects the ATP-bound state, with the two nucleotide-binding domains in close contact and the two transmembrane domains forming a central cavity--presumably the drug translocation pathway--that is shielded from the inner leaflet of the lipid bilayer and from the cytoplasm, but exposed to the outer leaflet and the extracellular space.