Universality of the Sodium Ion Binding Mechanism in Class A G‐Protein‐Coupled Receptors

The allosteric modulation of G‐protein‐coupled receptors (GPCRs) by sodium ions has received significant attention as crystal structures of several receptors show Na⁺ ions bound to the inactive conformations at the conserved Asp^2.50. To date, structures from 24 families of GPCRs have been determined, though mechanistic insights into Na⁺ binding to the allosteric site are limited. We performed hundreds‐of‐microsecond long simulations of 18 GPCRs and elucidated their Na⁺ binding mechanism. In class A GPCRs, the Na⁺ ion binds to the conserved residue 2.50 whereas in class B receptors, it binds at 3.43b, 6.53b, and 7.49b. Using Markov state models, we obtained the free energy profiles and kinetics of Na⁺ binding to the allosteric site, which reveal a conserved mechanism of Na⁺ binding for GPCRs and show the residues that act as major barriers for ion diffusion. Furthermore, we also show that the Na⁺ ion can bind to GPCRs from the intracellular side when the allosteric site is inaccessible from the extracellular side.     

Differences between G‐Protein‐Stabilized Agonist–GPCR Complexes and their Nanobody‐Stabilized Equivalents

Protein nanobodies have been used successfully as surrogates for unstable G‐proteins in order to crystallize G‐protein‐coupled receptors (GPCRs) in their active states. We used molecular dynamics (MD) simulations, including metadynamics enhanced sampling, to investigate the similarities and differences between GPCR–agonist ternary complexes with the α‐subunits of the appropriate G‐proteins and those with the protein nanobodies (intracellular binding partners, IBPs) used for crystallization. In two of the three receptors considered, the agonist‐binding mode differs significantly between the two alternative ternary complexes. The ternary‐complex model of GPCR activation entails enhancement of ligand binding by bound IBPs: Our results show that IBP‐specific changes can alter the agonist binding modes and thus also the criteria for designing GPCR agonists.     

A Usual G‐Protein‐Coupled Receptor in Unusual Membranes

G‐protein‐coupled receptors (GPCRs) are the largest family of membrane‐bound receptors and constitute about 50 % of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge‐interaction‐directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid‐ and polymer‐based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non‐biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H⁺] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin‐conserved ERY sequence motif that helps break the cytoplasmic “ionic lock”. This study unveils a novel design concept of non‐biological membranes to reconstitute and harness GPCR functions in synthetic systems.     

Stabilizer‐Guided Inhibition of Protein–Protein Interactions

The discovery of novel protein–protein interaction (PPI) modulators represents one of the great molecular challenges of the modern era. PPIs can be modulated by either inhibitor or stabilizer compounds, which target different though proximal regions of the protein interface. In principle, protein–stabilizer complexes can guide the design of PPI inhibitors (and vice versa). In the present work, we combine X‐ray crystallographic data from both stabilizer and inhibitor co‐crystal complexes of the adapter protein 14‐3‐3 to characterize, down to the atomic scale, inhibitors of the 14‐3‐3/Tau PPI, a potential drug target to treat Alzheimer’s disease. The most potent compound notably inhibited the binding of phosphorylated full‐length Tau to 14‐3‐3 according to NMR spectroscopy studies. Our work sets a precedent for the rational design of PPI inhibitors guided by PPI stabilizer–protein complexes while potentially enabling access to new synthetically tractable stabilizers of 14‐3‐3 and other PPIs.     

Nanobody‐Conjugated Nanotubes for Targeted Near‐Infrared In Vivo Imaging and Sensing

Fluorescent nanomaterials such as single‐walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP‐binding nanobodies to DNA‐wrapped SWCNTs. This approach combines the targeting capabilities of GFP‐binding nanobodies and the nonbleaching near‐infrared fluorescence (850–1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin‐5‐GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.     

Substrate Activity Screening with Kinases: Discovery of Small‐Molecule Substrate‐Competitive c‐Src Inhibitors

Substrate‐competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small‐molecule substrates for any protein kinase were discovered, as well as the first substrate‐competitive inhibitors of c‐Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate‐competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP‐competitive inhibitors.     

Allosteric Optical Control of a Class B G‐Protein‐Coupled Receptor

Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca²⁺, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R.     

Exploring Weak Ligand–Protein Interactions by Long‐Lived NMR States: Improved Contrast in Fragment‐Based Drug Screening

Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long‐lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N‐terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K_D. This property makes the LLS method particularly attractive for the initial steps of fragment‐based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.     

Structurally Defined αMHC‐II Nanobody–Drug Conjugates: A Therapeutic and Imaging System for B‐Cell Lymphoma

Antibody–drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full‐sized antibodies. Camelid‐derived single‐domain antibody fragments (VHHs or nanobodies) offer a possible solution to this challenge by providing expedited target screening and validation through switching between imaging and therapeutic activities. We used a nanobody (VHH7) specific for murine MHC‐II and rendered “sortase‐ready” for the introduction of oligoglycine‐modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial monoclonal antibodies (mAbs) for internalization and exhibited high specificity and cytotoxicity against A20 murine B‐cell lymphoma. Non‐invasive NIR imaging with a VHH7–fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody–drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity.