Arrestin-1 engineering facilitates complex stabilization with native rhodopsin

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).

Differential Regulation of GPCRs—Are GRK Expression Levels the Key?

“…Thus, GRK-mediated receptor phosphorylation is crucial for the formation of "core" and "hanging" GPCR-arrestin complexes. Phosphorylation is often also hypothesized to be the starting point of arrestin complex formation, however, the precise determination of succession of these binding events is still occluded, as arrestins also have an affinity for active, yet unphosphorylated GPCRs (Gurevich and Gurevich, 2006;Haider et al, 2019;Drube et al, 2021). Differential spacing of negative charges at the receptor C-terminus has been shown to induce specific conformational changes in arrestins (Lee et al, 2016;Nuber et al, 2016;Mayer et al, 2019).…”

New Insights into Arrestin Recruitment to GPCRs

“…To further validate the direct assay, we compared the recruitment efficacies of different β-arrestin-2 mutants to the B2AR in the direct assay and a classical BRET-based approach and compared the results obtained from both assays. The function of these arrestin mutants was described before [ 34 , 35 ]. No significant difference between the results of the two assays could be observed, providing evidence that the direct assay reports accurate measurements for relative binding strength ( Figure 3 b).…”

Biochemical insights into structure and function of arrestins

“…Their results also imply differential engagement of the flexible finger loop depending on the receptor activation state. Later on, Haider et al selected from the same library a subset of 57 Ala-arr-1 mutants and generated double, triple, and quadruple Ala mutants in a combinatorial manner and tested their affinity to different Rho states [66]. Mutants with a disrupted polar core and three-element interaction no longer preferred Rho*-P over inactive (dark-state) phosphorylated Rho (Rho-P) and active nonphosphorylated (Rho*) rhodopsin.…”