Abstract
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 (IC50) 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).
Highlights
Arrestins are most prominent interaction partners for G protein-coupled receptors (GPCRs) and play an essential role as signal-terminating proteins
Advances in creating a stable, high-affinity rhodopsin–arrestin complex have already been made by strategic mutagenesis[45], utilizing the accumulated knowledge of arrestin mutations gathered over the course of more than 20 years[44]
In this novel protein engineering approach, which is potentially applicable to any mutable binding partner of a GPCR, we demonstrated a dramatic increase in speed to create proteins with desired functionalities compared to traditional methods
Summary
Seven out of eleven double mutants that bore low expression levels, had IC50 values significantly higher than the F375A single mutant We presume that such mutations increase the flexibility of the arrestin molecule and thereby facilitate binding to rhodopsin, but make the protein prone to degradation due to low conformational stability or disturbance of the protein fold. Other mutations, such as F380A, showed a significantly higher expression level compared to WT arrestin-1 and were subsequently introduced into further mutant combinations, in the attempt to balance the functional expression and rhodopsin–arrestin mutant binding levels. Increasing levels of engineering reduces the sensitivity of arrestin-1 for binding to typically non-preferred receptor states, except for ROS
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
