Ned Van Eps

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein α subunit (Gα). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the α5 helix of Gα that would perturb GDP binding at the β6-α5 loop. This movement was not observed when a flexible loop was inserted between the α5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from Gα.

Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit

In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the Gα subunit of a heterotrimeric G protein. In an initial step, receptor interaction with Gα acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechanism is unknown. In this study, site-directed spin labeling and double electron–electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of Gα, the extension of which is a high-affinity receptor binding element.

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.

Structure and function of the visual arrestin oligomer

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter‐spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter‐subunit distance measurements revealed that only arrestin monomer binds to light‐activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark‐adapted photoreceptors. Thus, the tetramer likely serves as a ‘storage’ form of arrestin, increasing the arrestin‐binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.

Conformation of receptor-bound visual arrestin

Arrestin-1 (visual arrestin) binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate G-protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin-1 and double electron–electron resonance was used to monitor interspin distance changes upon P-Rh* binding. The results indicate that the relative position of the N and C domains remains largely unchanged, contrary to expectations of a “clam-shell” model. A loop implicated in P-Rh* binding that connects β-strands V and VI (the “finger loop,” residues 67–79) moves toward the expected location of P-Rh* in the complex, but does not assume a fully extended conformation. A striking and unexpected movement of a loop containing residue 139 away from the adjacent finger loop is observed, which appears to facilitate P-Rh* binding. This change is accompanied by smaller movements of distal loops containing residues 157 and 344 at the tips of the N and C domains, which correspond to “plastic” regions of arrestin-1 that have distinct conformations in monomers of the crystal tetramer. Remarkably, the loops containing residues 139, 157, and 344 appear to have high flexibility in both free arrestin-1 and the P-Rh*complex.

Activation of the A2A adenosine G-protein-coupled receptor by conformational selection

Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use 19F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as ‘ionic lock’) between transmembrane helices 3 and 6; and (2) two active states, S3 and S3′, as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3′, in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.

A Model for the Solution Structure of the Rod Arrestin Tetramer

Visual rod arrestin has the ability to self-associate at physiological concentrations. We previously demonstrated that only monomeric arrestin can bind the receptor and that the arrestin tetramer in solution differs from that in the crystal. We employed the Rosetta docking software to generate molecular models of the physiologically relevant solution tetramer based on the monomeric arrestin crystal structure. The resulting models were filtered using the Rosetta energy function, experimental intersubunit distances measured with DEER spectroscopy, and intersubunit contact sites identified by mutagenesis and site-directed spin labeling. This resulted in a unique model for subsequent evaluation. The validity of the model is strongly supported by model-directed crosslinking and targeted mutagenesis that yields arrestin variants deficient in self-association. The structure of the solution tetramer explains its inability to bind rhodopsin and paves the way for experimental studies of the physiological role of rod arrestin self-association.

Mapping allosteric connections from the receptor to the nucleotide-binding pocket of heterotrimeric G proteins

Heterotrimeric G proteins function as molecular relays that mediate signal transduction from heptahelical receptors in the cell membrane to intracellular effector proteins. Crystallographic studies have demonstrated that guanine nucleotide exchange on the Gα subunit causes specific conformational changes in three key “switch” regions of the protein, which regulate binding to Gβγ subunits, receptors, and effector proteins. In the present study, nitroxide side chains were introduced at sites within the switch I region of Gαi to explore the structure and dynamics of this region throughout the G protein cycle. EPR spectra obtained for each of the Gα(GDP), Gα(GDP)βγ heterotrimer and Gα(GTPγS) conformations are consistent with the local environment observed in the corresponding crystal structures. Binding of the heterotrimer to activated rhodopsin to form the nucleotide-free (empty) complex, for which there is no crystal structure, causes prominent changes relative to the heterotrimer in the structure of switch I and contiguous sequences. The data identify a putative pathway of allosteric changes triggered by receptor binding and, together with previously published data, suggest elements of a mechanism for receptor-catalyzed nucleotide exchange.

Structural and dynamical changes in an α-subunit of a heterotrimeric G protein along the activation pathway

The Gα subunits of heterotrimeric G proteins (Gαβγ) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in “switch” sequences of Gα, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gβγ subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Gαi1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix α4, a putative effector binding region. In the inactive Gαi1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gβγ, motions of R1 are highly constrained, reflecting direct contact interactions at the Gαi1–Gβ interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding α2/β4 loop and smaller allosteric changes at the Gαi1–Gβγ interface distant from the receptor binding surface. Upon addition of GTPγS, the activated Gαi1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in α4, distant from the interaction surface, supports a role for this helix in effector binding.