The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.     

F NMR studies of solvent exposure and peptide binding to an SH3 domain

¹⁹F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct ¹⁹F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. ¹⁹F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. ¹⁹F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Pro-diaminopropionic acid –NH₂, known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the ¹⁹F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 ± 4 μM was obtained. Analysis of the line widths suggested an average exchange rate, k_ex, associated with the peptide–protein two-site exchange, of 5200 ± 600 s^− 1 at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3–domain peptide complexes. These results show the potential utility of ¹⁹F-metafluorotyrosine to probe protein–protein interactions in conjunction with paramagnetic contrast agents.     

Structure and orientation of T4 lysozyme bound to the small heat shock protein alpha-crystallin

We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat shock protein α-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active-site cleft. Furthermore, increased distances between i and i + 4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment, while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, electron paramagnetic resonance spectral line shapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and α-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two modes of substrate binding originate from two discrete binding sites on the chaperone.     

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.     

Residue specific partitioning of KL4 into phospholipid bilayers

KL₄, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL₄ and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL₄ on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL₄, and the partitioning depths of specific KL₄ residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL₄ induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL₄ (V.C. Antharam et al., 2009), we are able to distinguish how KL₄ affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL₄ also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL₄ are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.