Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

The G protein‐coupled adenosine A_2A receptor (A_2AAR) is an important new (potential) drug target in immuno‐oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A_2AAR antagonists displaying exceptional selectivity. While crystal structures of the human A_2AAR have been solved, mostly using the A_2A‐StaR2 protein that bears 9 point mutations, co‐crystallization with Preladenant derivatives has so far been elusive. We developed a new A_2AAR construct harboring a single point mutation (S91^3.39K) which renders it extremely thermostable. This allowed the co‐crystallization of two novel Preladenant derivatives, the polyethylene glycol‐conjugated (PEGylated) PSB‐2113, and the fluorophore‐labeled PSB‐2115. The obtained crystal structures (2.25 Å and 2.6 Å resolution) provide explanations for the high potency and selectivity of Preladenant derivatives. They represent the first crystal structures of a GPCR in complex with PEG‐ and fluorophore‐conjugated ligands. The applied strategy is predicted to be applicable to further class A GPCRs.

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Par-4 is a unique proapoptotic protein with the ability to induce apoptosis selectively in cancer cells. The X-ray crystal structure of the C-terminal domain of Par-4 (Par-4_CC), which regulates its apoptotic function, was obtained by MAD phasing. Par-4 homodimerizes by forming a parallel coiled-coil structure. The N-terminal half of Par-4_CC contains the homodimerization subdomain. This structure includes a nuclear export signal (Par-4_NES) sequence, which is masked upon dimerization indicating a potential mechanism for nuclear localization. The heteromeric-interaction models specifically showed that charge interaction is an important factor in the stability of heteromers of the C-terminal leucine zipper subdomain of Par-4 (Par-4_LZ). These heteromer models also displayed NES masking capacity and therefore the ability to influence intracellular localization.

Crosstalk between opioid and adrenergic receptors is well characterized and due to interactions between second messenger systems, formation of receptor heterodimers, and extracellular allosteric binding regions. Both classes of receptors bind both sets of ligands. We propose here that receptor crosstalk may be mirrored in ligand complementarity. We demonstrate that opioids bind to adrenergic compounds with micromolar affinities. Additionally, adrenergic compounds bind with micromolar affinities to extracellular loops of opioid receptors while opioids bind to extracellular loops of adrenergic receptors. Thus, each compound type can bind to the complementary receptor, enhancing the activity of the other compound type through an allosteric mechanism. Screening for ligand complementarity may permit the identification of other mutually-enhancing sets of compounds as well as the design of novel combination drugs or tethered compounds with improved duration and specificity of action.