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Termination of the G-protein-coupled receptor signaling involves phosphorylation of its C-terminus and subsequent binding of the regulatory protein arrestin. In the visual system, arrestin-1 preferentially binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction. Here, we have investigated binding of a synthetic phosphopeptide of bovine rhodopsin (residues 323–348) to the active variants of visual arrestin-1: splice variant p44, and the mutant R175E. Unlike the wild type arrestin-1, both these arrestins are monomeric in solution. Solution structure analysis using small angle X-ray scattering supported by size exclusion chromatography results reveal dimerization in both the arrestins in the presence of phosphopeptide. Our results are the first report, to our knowledge, on receptor-induced oligomerization in arrestin, suggesting possible roles for the cellular function of arrestin oligomers. Given high structural homology and the similarities in their activation mechanism, these results are expected to have implications for all arrestin isoforms. [ABSTRACT FROM AUTHOR]

Human guanylate‐binding protein 1 (hGBP1) belongs to the family of dynamin‐like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post‐translationally modified by attachment of a farnesyl group yielding farn‐hGBP1. In this study, hydrodynamic differences in farn‐hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size‐exclusion chromatography (SEC). In addition, we performed small‐angle X‐ray scattering (SAXS) experiments coupled with a SEC setup (SEC‐SAXS) to investigate structural properties of nonmodified hGBP1 and farn‐hGBP1 in solution. SEC‐SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal‐like conformation in nucleotide‐free solution, whereas unmodified hGBP1 forms a monomer–dimer equilibrium both in the inactive ground state in nucleotide‐free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn‐hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn‐hGBP1 that occur in the presence of nucleotides. Database: All SEC‐SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8. [ABSTRACT FROM AUTHOR]

Background: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime ofthe adduct state. Results: Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine a-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Â resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the ß-scaffold ofthe core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Ca backbone of ß strands in the N- and C-terminal ends of the LOV core domain. Conclusions: The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria. [ABSTRACT FROM AUTHOR]

Light–Oxygen–Voltage (LOV) domains represent the photo-responsive domains of various blue-light photoreceptor proteins and are widely distributed in plants, algae, fungi, and bacteria. Here, we report the dark-state crystal structure of PpSB1-LOV, a slow-reverting short LOV protein from Pseudomonas putida that is remarkably different from our previously published “fully light-adapted” structure [1]. A direct comparison of the two structures provides insight into the light-activated signaling mechanism. Major structural differences involve a ~ 11 Å movement of the C terminus in helix Jα, ~ 4 Å movement of Hβ–Iβ loop, disruption of hydrogen bonds in the dimer interface, and a ~ 29° rotation of chain-B relative to chain-A as compared to the light-state dimer. Both crystal structures and solution NMR data are suggestive of the key roles of a conserved glutamine Q116 and the N-cap region consisting of A′α–Aβ loop and the A′α helix in controlling the light-activated conformational changes. The activation mechanism proposed here for the PpSB1-LOV supports a rotary switch mechanism and provides insights into the signal propagation mechanism in naturally existing and artificial LOV-based, two-component systems and regulators. [ABSTRACT FROM AUTHOR]