Arrestin-1 engineering facilitates complex stabilization with native rhodopsin

• Published in: Scientific reports 2019-01, Vol.9 (1), p.439-439, Article 439
• Main Authors: Haider, Raphael S., Wilhelm, Florian, Rizk, Aurélien, Mutt, Eshita, Deupi, Xavier, Peterhans, Christian, Mühle, Jonas, Berger, Philipp, Schertler, Gebhard F. X., Standfuss, Jörg, Ostermaier, Martin K.
• Format: Article
• Language: English
• Subjects: 38; 631/45/612/194; 631/535; 692/699/3161/3175; 82; 82/80; 82/83; 96; 96/106; 96/109; 96/35; Arrestin; G protein-coupled receptors; Humanities and Social Sciences; Light; multidisciplinary; Mutants; Mutation; Phosphorylation; Proteins; Retina; Rhodopsin; Science; Science (multidisciplinary); Sodium chloride
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-36881-4

Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin−arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC 50 ) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin–arrestin-1 complex. The achieved stabilization of the active rhodopsin–arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).