Conditional and Reversible Activation of Class A and B G Protein-Coupled Receptors Using Tethered Pharmacology

Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of ExONatide, a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. ExONatide covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca2+ rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent beta-mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using GhrelON, a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, ExONatide and GhrelON allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both in vitro and in vivo, with applicability across GPCRs

LanM Peptides – Unravelling the Binding Properties of the EF-Hand Loop Sequences Stripped from the Structural Corset

Since the discovery of the biological relevance of lanthanides (Lns) for methylotrophic bacteria in the last decade, the field has seen a steady rise in discoveries of bacteria using Lns. The major role of lanthanides here is in the active sites of enzymes: methanol dehydrogenases. Additionally, lanthanide binding proteins have also been identified. One such protein is lanmodulin (LanM) and, with a remarkable selectivity for Lns over Ca(II) and affinities in the picomolar range, it makes an attractive target to address challenges in lanthanide separation. Why LanM has such a high selectivity is currently not entirely understood, both the specific amino acid sequences of the EF-hand loops, together with cooperativity effects have been suggested. Consequently, we decided to remove the effect of cooperativity by focusing on the amino acid level. Thus, we synthesized all four 12-amino acid EF-Hand loop peptides of LanM using solid phase peptide synthesis and investigated their affinity for Lns (Eu(III), Tb(III)), the actinide Cm(III) and Ca(II). Using isothermal titration calorimetry and time resolved laser fluorescence spectroscopy combined with parallel factor analysis, we show that in the absence of cooperativity the short EF-Hand loop peptides have all similar affinities for lanthanides and that these are all in the micromolar range. Furthermore, calcium was shown not to bind to the peptides which was verified with circular dichroism spectroscopy. This technique also revealed that the peptides undergo a change to a more ordered state when lanthanides are added. These experimental observations were further supported by molecular dynamics simulations. Lastly, we put Eu(III) and Cm(III) in direct competition using TRLFS. Remarkably, a slightly higher affinity for the actinide, as was also observed for LanM, was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring in the full protein and therefore reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium

Data publication: Lanmodulin Peptides – Unravelling the Binding of the EF-Hand Loop Sequences Stripped from the Structural Corset

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