Sergey A. Vishnivetskiy

For many applications, including structural, biochemical, and biophysical studies, proteins must be expressed in heterologous systems and purified. Expression in cell-free systems avoids several pi...

In all arrestins, the gate loop is the central part of the lariat loop, which has an unusual shape and participates in maintaining the basal conformation. The gate loop supplies two out of five charges that constitute a stabilizing intramolecular interaction, aspartates in the polar core between the two domains. To elucidate the functional role of individual gate loop residues, we performed comprehensive site-directed mutagenesis and tested the effects of mutations on arrestin-1 binding to its preferred target, phosphorylated light-activated rhodopsin, and unphosphorylated activated form. Out of 34 mutations tested, 24 and 25 affected the binding to phosphorylated and unphosphorylated rhodopsin, respectively. Manipulation of residues following polar core aspartates reduced preference for phosphorylated over unphosphorylated light-activated rhodopsin as dramatically as replacing these negatively charged aspartates with positively charged arginine. The data show that numerous lariat loop residues play distinct roles in arrestin-1 binding and its exquisite preference for phosphorylated light-activated rhodopsin.

In all arrestins the gate loop is the central part of the lariat loop, which has an unusual shape and participates in maintaining the basal conformation. Gate loop supplies two out of five charges that constitute a stabilizing intramolecular interaction, the polar core between the two domains. To elucidate the functional role of individual gate loop residues we performed comprehensive site-directed mutagenesis and tested the effects of mutations on arrestin-1 binding to its preferred target, phosphorylated light-activated rhodopsin, and unphosphorylated activated form. Out of 34 mutations tested, 24 and 25 affected the binding to phosphorylated and unphosphorylated rhodopsin, respectively. Manipulation of residues following polar core aspartates reduced preference for phosphorylated over unphosphorylated light-activated rhodopsin as dramatically as replacing these negatively charged aspartates with positively charged arginine. The data show that numerous lariat loop residues play distinct roles in arrestin-1 binding and its exquisite preference for phosphorylated light-activated rhodopsin.

Sequences and three-dimensional structures of the four vertebrate arrestins are very similar, yet in sharp contrast to other subtypes, arrestin-1 demonstrates exquisite selectivity for the active phosphorylated form of its cognate receptor, rhodopsin. The N-terminus participates in receptor binding and serves as the anchor of the C-terminus, the release of which facilitates arrestin transition into a receptor-binding state. We tested the effects of substitutions of fourteen residues in the N-terminus of arrestin-1 on the binding to phosphorylated and unphosphorylated light-activated rhodopsin of wild-type protein and its enhanced mutant with C-terminal deletion that demonstrates higher binding to both functional forms of rhodopsin. Profound effects of mutations identified lysine-15 as the main phosphate sensor and phenylalanine-13 as the key anchor of the C-terminus. These residues are conserved in all arrestin subtypes. Substitutions of five other residues reduced arrestin-1 selectivity for phosphorylated rhodopsin, indicating that wild-type residues participate in fine-tuning of arrestin-1 binding. Differential effects of numerous substitutions in wild-type and an enhanced mutant arrestin-1 suggest that these two proteins bind rhodopsin differently.

Sequences and three-dimensional structures of the four vertebrate arrestins are very similar, yet in sharp contrast to other subtypes arrestin-1 demonstrates exquisite selectivity for the active phosphorylated form of its cognate receptor, rhodopsin. The N-terminus participates in receptor binding and serves as the anchor of the C-terminus, the release of which facilitates arrestin transition into receptor-binding state. We tested the effects of substitutions of fourteen residues in the N-terminus of arrestin-1 on the binding to phosphorylated and unphosphorylated light-activated rhodopsin of wild type protein and its enhanced mutant with C-terminal deletion that demonstrates higher binding to both functional forms of rhodopsin. Profound effects of mutations identified lysine-15 as the main phosphate sensor and phenylalanine-13 as the key anchor of the C-terminus. These residues are conserved in all arrestin subtypes. Substitutions of five other residues reduced arrestin-1 selectivity, indicating that wild type residues participate in fine-tuning of arrestin- 1 binding to rhodopsin. Differential effects of numerous substitutions in wild type and an enhanced mutant arrestin-1 shows that these two proteins bind rhodopsin differently.

Arrestin-1, or visual arrestin, exhibits an exquisite selectivity for light-activated phosphorylated rhodopsin (P-Rh*) over its other functional forms. That selectivity is believed to be mediated by two well-established structural elements in the arrestin-1 molecule, the activation sensor detecting the active conformation of rhodopsin and the phosphorylation sensor responsive to the rhodopsin phosphorylation, which only active phosphorylated rhodopsin can engage simultaneously. However, in the crystal structure of the arrestin-1–rhodopsin complex there are arrestin-1 residues located close to rhodopsin, which do not belong to either sensor. Here we tested by site-directed mutagenesis the functional role of these residues in wild type arrestin-1 using a direct binding assay to P-Rh* and light-activated unphosphorylated rhodopsin (Rh*). We found that many mutations either enhanced the binding only to Rh* or increased the binding to Rh* much more than to P-Rh*. The data suggest that the native residues in these positions act as binding suppressors, specifically inhibiting the arrestin-1 binding to Rh* and thereby increasing arrestin-1 selectivity for P-Rh*. This calls for the modification of a widely accepted model of the arrestin–receptor interactions.

Arrestin-1, or visual arrestin, exhibits an exquisite selectivity for light-activated phosphorylated rhodopsin (P-Rh*) over its other functional forms. That selectivity is believed to be mediated by two well-established structural elements in the arrestin-1 molecule, the activation sensor detecting the active conformation of rhodopsin and the phosphorylation sensor responsive to the rhodopsin phosphorylation, which only active phosphorylated rhodopsin can engage simultaneously. However, in the crystal structure of the arrestin-1-rhodopsin complex there are arrestin-1 residues located close to rhodopsin, which do not belong to either sensor. Here we tested by site-directed mutagenesis the functional role of these residues in wild type arrestin-1 using direct binding assay to P-Rh* and light-activated unphosphorylated rhodopsin (Rh*). We found that many mutations either enhanced the binding only to Rh* or increased the binding to Rh* much more than to P-Rh*. The data suggest that the native residues in these positions act as binding suppressors, specifically inhibiting the arrestin-1 binding to Rh* and thereby increasing arrestin-1 selectivity for P-Rh*. This calls for the modification of a widely accepted model of the arrestin-receptor interactions.

Arrestins preferentially bind active phosphorylated G protein-coupled receptors (GPCRs). The middle loop, highly conserved in all arrestin subtypes, is localized in the central crest on the GPCR-binding side. Upon receptor binding, it directly interacts with bound GPCR and demonstrates the largest movement of any arrestin element in the structures of the complexes. Comprehensive mutagenesis of the middle loop of rhodopsin-specific arrestin-1 suggests that it primarily serves as a suppressor of binding to non-preferred forms of the receptor. Several mutations in the middle loop increase the binding to unphosphorylated light-activated rhodopsin severalfold, which makes them candidates for improving enhanced phosphorylation-independent arrestins. The data also suggest that enhanced forms of arrestin do not bind GPCRs exactly like the wild-type protein. Thus, the structures of the arrestin-receptor complexes, in all of which different enhanced arrestin mutants and reengineered receptors were used, must be interpreted with caution.

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.

G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction.