Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. Here we report the first crystal structure of arrestin-3, solved at 3.0Å. Arrestin-3 is an elongated two-domain molecule with the overall fold and key inter-domain interactions that hold free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and -3 we show that the presence of this loose structure correlates with reduced arrestin selectivity for activated receptor, consistent with a conformational change in this β-sheet upon receptor binding.

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NaChBac, a six-␣-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na ؉ -selective channel (Ren, D., Navarro, B., Xu, H., Yue, L., Shi, Q., and Clapham, D. E. (2001) Science 294, 2372-2375). As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na ؉ channels (Na V PZ from Paracoccus zeaxanthinifaciens and Na V SP from Silicibacter pomeroyi). Na V PZ and Na V SP share ϳ40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na V PZ and Na V SP were Na ؉ -selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-␣-helical transmembranespanning subunits constitute a widely distributed superfamily (Na V Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na V Bac structure and function of similarity and dissimilarity among Na V Bac proteins. Thus, the Na V Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.Mammalian voltage-gated sodium (Na V ) 1 and calcium (Ca V ) channels underlie membrane excitability, muscle contraction, and hormone secretion (1). In contrast, the function of prokaryotic voltage-gated ion-selective channels is relatively unknown. Na ϩ channels may drive Na ϩ -dependent flagellar motors in certain marine and alkaliphilic species (2-6). In marine vibrio, PomAB and MotXY have been proposed to form a functional Na ϩ channel (5, 6), but the conductance has not been directly measured. In alkaliphilic bacteria, the prokaryotic ion channel responsible has not been identified. A bacterial 6-␣-helical transmembrane (6TM) channel subunit NaChBac was expressed in CHO cells as a functional voltage-gated Na ϩ channel (7), but its role in bacteria is still being elucidated.The pore-forming subunits (␣ 1 ) of mammalian Na V and Ca V are composed of four similar repeats of 6TM domains (8, 9), probably arising by gene duplication of a single 6TM gene (1, 10). The first bacterial voltage-gated Na ϩ channel (NaChBac) functionally expressed in mammalian cells was cloned from Bacillus halodurans (7). It contains a single 6TM domain of 274 amino acids but almost certainly forms a tetramer (7). NaChBac voltagedependent activation and inactivation kinetics are 10 -100 times slower than that of Na V (7). NaChBac inactivation may result from pore inactivation (C-type inactivation), since it does not contain an obvious cytoplasmic inactivation gate (7).Because single 6TM Na ϩ -selective ion channels do not appear to be present in vertebrates, the 24 TM structure of Na V s may have arisen under evolut...