Surveying nonvisual arrestins reveals allosteric interactions between functional sites

Abstract

Arrestins are important scaffolding proteins that are expressed in all vertebrate animals. They regulate cell-signaling events upon binding to active G-protein coupled receptors (GPCR) and trigger endocytosis of active GPCRs. While many of the functional sites on arrestins have been characterized, the question of how these sites interact is unanswered. We used anisotropic network modeling (ANM) together with our covariance compliment techniques to survey all the available structures of the nonvisual arrestins to map how structural changes and protein-binding affect their structural dynamics. We found that activation and clathrin binding have a marked effect on arrestin dynamics, and that these dynamics changes are localized to a small number of distant functional sites. These sites include α-helix 1, the lariat loop, nuclear localization domain, and the C-domain β-sheets on the C-loop side. Our techniques suggest that clathrin binding and/or GPCR activation of arrestin perturb the dynamics of these sites independent of structural changes.

Keywords: GPCR; allostery; anisotropic network modeling; arrestin.

Figure 1:

The A) domain and B) crystallography structure of Arrestin-2. The N-domain (blue), C-domain (orange), and C-tail domain (red) are shown along with the polar core domain (spheres). The discontinuity in the red backbone exists because a ~35-residue region of the C-tail domain is missing from all crystal structures.

Figure 2:

Changes in the structure of arrestin upon activation. Inactive arrestin (solid) and active arrestin (translucent) are superimposed on touch of each other, with the N-terminal domain on the left as in Figure 1. The polar core (spheres), lariat loop (purple), GPCR (green), and GPCR C-terminal peptide (green sticks) are shown. Blue and purple regions represent sequence which is included in our ANM modelling. Arrestin activation is marked by the disruption of the polar core residues (top left panel), and a rotation of the C-domain with respect to the N-domain (top right panel).

Figure 3:

A pairwise comparison of the A) root mean squared deviation (RMSD) and B) the covariance complement derived from the 26 different structures analyzed in this study. Structures are color coded both by their ligands (left and bottom of graph), or whether they are arrestin-2 (gray) or arrestin-3 (red) (right of graph).

Figure 4:

A pairwise dot product of the first and second modes of motion as computed by ANM for all 26 structures analyzed. Structures are color coded both by their ligands (left and bottom of graph), or whether they are arrestin-2 (gray) or arrestin-3 (red) (right of graph).

Figure 5:

Average difference in the per-residue dot product between the first mode of all unliganded and peptide-bound structures for Arrestin-2 (A) or Arrestin-3 (B). Gray regions represent structural elements not included in the ANM model. Blue regions represent regions which did not contribute to the differences in the dot product, orange regions represent regions of maximal contribution.

Figure 6:

Average difference in the per-residue dot product between the first mode of all unliganded and clathrin-bound Arrestin-2. Gray regions represent structural elements not included in the ANM model. Blue regions represent regions which did not contribute to the differences in the dot product, orange regions represent regions of maximal contribution.