Journal of Molecular Biology
CommunicationCrystal Structure of p44, a Constitutively Active Splice Variant of Visual Arrestin
Graphical Abstract
Highlights
► The crystal structure of the arrestin splice variant p44 was determined at a resolution of 1.85 Å. ► Structural differences in flexible loop V–VI and polar core regions are highlighted. ► The electrostatic potential is remarkably positive on the N-domain and the C-domain in p44. ► The p44 structure provides the first direct structural evidence supporting the role of C-tail in the arrestin activation mechanism. ► The p44 structure represents an active conformation..
Introduction
Arrestins are regulatory proteins that play a central role in the termination of signal transduction pathways mediated by G-protein-coupled receptors, which are utilized by a wide variety of eukaryotic organisms to communicate environmental signals. Inactivation of a G-protein-coupled receptor involves a two-step mechanism: rapid phosphorylation, followed by binding of arrestin. The latter step sterically blocks further interaction of G-protein with the receptor, terminating the primary signaling event.1
Visual arrestin arr-1 is one of the most abundant soluble proteins in vertebrate photoreceptors that was originally isolated from bovine retina.2 Since then, several homologues of arrestin have been identified in a large number of species and nonretinal tissues. Among mammalian arrestin members, visual arrestins arr-1 and arr-4 are expressed at high levels in rod and cone photoreceptors, respectively. β-Arr-1 and β-arr-2 belong to a subfamily of ubiquitously expressed nonvisual arrestins and are involved in the inactivation of β-adrenergic receptors.3 Additionally, splice variants of arrestin have been reported previously for rod-arrestin and β-arrestin.4, 5, 6, 7, 8, 9 Each of the splice variants is derived from alternative mRNA splicing at the 3′ end, producing truncated isoforms. A splice variant of arr-1, termed p44, differs from arr-1 in three properties: the amino acid sequence is identical except for the C-terminus, where residues 371–404 are missing and Phe370 is replaced by Ala; it shows a high affinity for different rhodopsin species [light-activated rhodopsin (Rh⁎), light-activated phosphorylated rhodopsin (P-Rh⁎), phosphorylated rhodopsin (P-Rh), and C-terminally truncated rhodopsin that lacks the potential sites of phosphorylation]; and it is permanently localized in rod outer segments (ROS).7, 10 Recently, a 50-kDa variant of arr-1 was identified in bovine ROS, which binds to rhodopsin in a light-independent manner.8 Both splice variants differ in their biochemical properties from arr-1, which binds specifically to P-Rh⁎ only.
Crystal structures of visual arrestins and β-arrestins have been reported previously in their basal states.3, 11, 12 Although visual arrestins and β-arrestins display apparent selectivity for their respective receptors, the overall fold is conserved among all subtypes. Generally, the structure comprises two domains of anti-parallel β-sheets with an unusual polar core embedded between the two domains. Electrostatic interactions in polar core residues are partly stabilized by the C-terminal tail in extended conformation, locking the molecule in its basal (inactive) state. In the present article, we report the crystal structure of recombinant p44 expressed in Saccharomyces cerevisiae. A detailed comparison with arr-1 reveals a conserved arrestin fold with significant differences in flexible loop regions and in the polar core, as well as a remarkably positive electrostatic potential for both N-domain and C-domain. Both residues in these regions and the electrostatic potential play an important role in receptor binding.1 In addition, the structure of p44 (a constitutively active splice variant of arrestin) provides the first direct structural insights supporting a role of the C-terminus and the polar core region in arrestin activation mechanism.
Section snippets
Crystal structure of p44
The statistics of X-ray data collection and refinement are documented in Table 1. The final monomer model comprises residues 10–360 of the protein. At the N-terminus and C-terminus, residues 1–9 and 361–370 could not be traced in the electron density map and thus are likely to be disordered. Additionally, no electron density was observed for loop V–VI (residue range 70–75). According to Ramachandran plots generated with MolProbity (PHENIX), the model exhibits good geometry, with none of the
Structural differences between p44 and arr-1
The crystal structure of arr-1 reported previously shows two different conformations among the four molecules present in the asymmetric unit, where two chains are in α-conformation and the other two chains are in β-conformation.11, 12 In the following sections, we compare the p44 model with the two arr-1 conformers [see Supplementary Information for Protein Data Bank (PDB) ID: 3UGX]. When superimposed, all three structures show the same fold (Fig. 1). The RMSD of both conformers α and β with
The molecular mechanism of arrestin activation
The crystal structures of visual arrestin determined so far are in basal or ‘inactive’ states and require activation by the binding of the phosphorylated carboxy-terminal segment of rhodopsin (first trigger step) (Fig. 4).1 Binding of the phosphoresidues of rhodopsin causes the displacement of the C-tail of arrestin, where the latter sterically blocks the polar core region. Although much of the C-terminus is flexible in arr-1 structures, the trigger might involve the disruption of
Accession codes
Coordinates and structure factors for arr-1 and p44 have been deposited in the PDB under accession codes 3UGX and 3UGU.
Acknowledgements
We are grateful to the beamline scientists at the European Synchrotron Radiation Facility (Grenoble, France) for providing assistance with the use of beamline ID14-4. We thank Oliver H. Weiergräber for comments on the manuscript, Bianca Krafft for generating the p44 clone in S. cerevisiae, and Dieter Willbold and the Russian Group ONEXIM (GB) for generous support.
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