Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Poster Abstracts

18-POS Board 18 SMALL MOLECULE MODULATION OF ARRESTIN BINDING TO G PROTEIN COUPLED RECEPTORS AT ARRESTIN-ISOFORM SPECIFICITY Han Kurt 1 ; Ali Akyol 3 ; Zeynep N Cinviz 2 ; Cagdas Son 3 ; Chen Zheng 4 ; Irene Gado 5 ; Massimiliano Meli 6 ; VSEVOLD V. GUREVICH 4 ; Giulia Morra 6 ; Ivan Bassanini 6 ; Erica Ferrandi 6 ; Francesca Vasile 5 ; Ozge Sensoy 2 ;

1 Universita degli Studi di Cagliari, Cagliari, Italy 2 Istanbul Medipol University, Istanbul, Turkey 3 The Middle East Technical University, Ankara , Turkey 4 Vanderbilt University, Nashville, TN, USA 5 University of Milano, Milano, Italy 6 SCITEC, Milano, Italy

G protein-coupled receptor (GPCR) signaling is terminated by binding of arrestin to activated and phosphorylated receptor. Nevertheless, phosphorylatable residues of the receptor might undergo mutation. Consequently, this precludes receptor desensitization and leads to excessive signaling, which underlies a variety of human disorders. Pre-activated arrestin mutants have been constructed for enabling phosphorylation-independent receptor binding, yet they turned out to be genetically unstable.We performed extensive coarse-grained (CG) molecular dynamics (MD) simulations of wild type arrestin-3 in water and at membrane by fine-tuning default parameters in Martini 3 force field and demonstrated that arrestin-3 sampled transiently pre-activated conformation. Here, we hypothesize that pre-activated conformation can be stabilized by small molecules, and phosphorylation-independent binding can be maintained. Towards this end, we did pocket search on the backmapped pre-activated conformation of arrestin-3 and identified a druggable pocket near the back loop. We discovered a compound that favorably binds to the back loop in the unfolded state and stabilizes the pre-activated conformation in microsecond long atomistic simulations. Saturation-transfer difference NMR data showed that the compound binds at the back loop of arrestin-3. Importantly, we showed that the compound, but not the others that bind to the back loop in the folded state, increased in-cell arrestin-3 binding to basal beta2-adrenergic receptor and its phosphorylation-deficient mutant as shown by FRET- and NanoBiT-based assays in living cells. We demonstrated by MD simulation that the compound did not stably bind to arrestin-2 and did not increase binding in-cell. Moreover, the binding is receptor specific as the compound did not increase binding of arrestin-3 to muscarinic M2 receptor, whose phosphorylation-dependency for arrestin binding is lower than that of beta2 adrenergic receptor. These experiments demonstrated the feasibility of enhancing binding of endogenous wild type arrestin-3 to GPCRs in a receptor-specific and arrestin-subtype selective manner via small molecules.

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