Carsten Schubert

A Model for Arrestin’s Regulation: The 2.8 Å Crystal Structure of Visual Arrestin

G protein-coupled signaling is utilized by a wide variety of eukaryotes for communicating information from the extracellular environment. Signal termination is achieved by the action of the arrestins, which bind to activated, phosphorylated G protein-coupled receptors. We describe here crystallographic studies of visual arrestin in its basal conformation. The salient features of the structure are a bipartite molecule with an unusual polar core. This core is stabilized in part by an extended carboxy-terminal tail that locks the molecule into an inactive state. In addition, arrestin is found to be a dimer of two asymmetric molecules, suggesting an intrinsic conformational plasticity. In conjunction with biochemical and mutagenesis data, we propose a molecular mechanism by which arrestin is activated for receptor binding.

Crystal structure of beta-arrestin at 1.9 A: possible mechanism of receptor binding and membrane Translocation.

BACKGROUND Arrestins are responsible for the desensitization of many sequence-divergent G protein-coupled receptors. They compete with G proteins for binding to activated phosphorylated receptors, initiate receptor internalization, and activate additional signaling pathways. RESULTS In order to understand the structural basis for receptor binding and arrestins function as an adaptor molecule, we determined the X-ray crystal structure of two truncated forms of bovine beta-arrestin in its cytosolic inactive state to 1.9 A. Mutational analysis and chimera studies identify the regions in beta-arrestin responsible for receptor binding specificity. beta-arrestin demonstrates high structural homology with the previously solved visual arrestin. All key structural elements responsible for arrestins mechanism of activation are conserved. CONCLUSIONS Based on structural analysis and mutagenesis data, we propose a previously unappreciated part in beta-arrestins mode of action by which a cationic amphipathic helix may function as a reversible membrane anchor. This novel activation mechanism would facilitate the formation of a high-affinity complex between beta-arrestin and an activated receptor regardless of its specific subtype. Like the interaction between beta-arrestins polar core and the phosphorylated receptor, such a general activation mechanism would contribute to beta-arrestins versatility as a regulator of many receptors.

The 2.8 A crystal structure of visual arrestin: a model for arrestin's regulation.

G protein-coupled signaling is utilized by a wide variety of eukaryotes for communicating information from the extracellular environment. Signal termination is achieved by the action of the arrestins, which bind to activated, phosphorylated G protein-coupled receptors. We describe here crystallographic studies of visual arrestin in its basal conformation. The salient features of the structure are a bipartite molecule with an unusual polar core. This core is stabilized in part by an extended carboxy-terminal tail that locks the molecule into an inactive state. In addition, arrestin is found to be a dimer of two asymmetric molecules, suggesting an intrinsic conformational plasticity. In conjunction with biochemical and mutagenesis data, we propose a molecular mechanism by which arrestin is activated for receptor binding.

HOW DOES ARRESTIN RESPOND TO THE PHOSPHORYLATED STATE OF RHODOPSIN

Visual arrestin quenches light-induced signaling by binding to light-activated, phosphorylated rhodopsin (P-Rh*). Here we present structure-function data, which in conjunction with the refined crystal structure of arrestin (Hirsch, J. A., Schubert, C., Gurevich, V. V., and Sigler, P. B. (1999)Cell, in press), support a model for the conversion of a basal or “inactive” conformation of free arrestin to one that can bind to and inhibit the light activated receptor. The trigger for this transition is an interaction of the phosphorylated COOH-terminal segment of the receptor with arrestin that disrupts intramolecular interactions, including a hydrogen-bonded network of buried, charged side chains, referred to as the “polar core.” This disruption permits structural adjustments that allow arrestin to bind to the receptor. Our mutational survey identifies residues in arrestin (Arg175, Asp30, Asp296, Asp303, Arg382), which when altered bypass the need for the interaction with the receptor’s phosphopeptide, enabling arrestin to bind to activated, nonphosphorylated rhodopsin (Rh*). These mutational changes disrupt interactions and substructures which the crystallographic model and previous biochemical studies have shown are responsible for maintaining the inactive state. The molecular basis for these disruptions was confirmed by successfully introducing structure-based second site substitutions that restored the critical interactions. The nearly absolute conservation of the mutagenically sensitive residues throughout the arrestin family suggests that this mechanism is likely to be applicable to arrestin-mediated desensitization of most G-protein-coupled receptors.

An Additional Phosphate-binding Element in Arrestin Molecule IMPLICATIONS FOR THE MECHANISM OF ARRESTIN ACTIVATION

Arrestins quench the signaling of a wide variety of G protein-coupled receptors by virtue of high-affinity binding to phosphorylated activated receptors. The high selectivity of arrestins for this particular functional form of receptor ensures their timely binding and dissociation. In a continuing effort to elucidate the molecular mechanisms responsible for arrestins selectivity, we used the visual arrestin model to probe the functions of its N-terminal β-strand I comprising the highly conserved hydrophobic element Val-Ile-Phe (residues 11–13) and the adjacent positively charged Lys14 and Lys15. Charge elimination and reversal in positions 14 and 15 dramatically reduce arrestin binding to phosphorylated light-activated rhodopsin (P-Rh*). The same mutations in the context of various constitutively active arrestin mutants (which bind to P-Rh*, dark phosphorylated rhodopsin (P-Rh), and unphosphorylated light-activated rhodopsin (Rh*)) have minimum impact on P-Rh* and Rh* binding and virtually eliminate P-Rh binding. These results suggest that the two lysines “guide” receptor-attached phosphates toward the phosphorylation-sensitive trigger Arg175 and participate in phosphate binding in the active state of arrestin. The elimination of the hydrophobic side chains of residues 11–13 (triple mutation V11A, I12A, and F13A) moderately enhances arrestin binding to P-Rh and Rh*. The effects of triple mutation V11A, I12A, and F13A in the context of phosphorylation-independent mutants suggest that residues 11–13 play a dual role. They stabilize arrestins basal conformation via interaction with hydrophobic elements in arrestins C-tail and α-helix I as well as its active state by interactions with alternative partners. In the context of the recently solved crystal structure of arrestins basal state, these findings allow us to propose a model of initial phosphate-driven structural rearrangements in arrestin that ultimately result in its transition into the active receptor-binding state.

Visual Arrestin Activity May Be Regulated by Self-association*

Visual arrestin is the protein responsible for rapid quenching of G-protein-coupled receptor signaling. Arrestin exists as a latent inhibitor which must be ‘activated’ upon contact with a phosphorylated receptor. X-ray crystal structures of visual arrestin exhibit a tetrameric arrangement wherein an asymmetric dimer with an extensive interface between conformationally different subunits is related to a second asymmetric dimer by a local two-fold rotation axis. To test the biological relevance of this molecular organization in solution, we carried out a sedimentation equilibrium analysis of arrestin at both crystallographic and physiological protein concentrations. While the tetrameric form can exist at the high concentrations used in crystallography experiments, we find that arrestin participates in a monomer/dimer equilibrium at concentrations more likely to be physiologically relevant. Solution interaction analysis of a proteolytically modified, constitutively active form of arrestin shows diminished dimerization. We propose that self-association of arrestin may provide a mechanism for regulation of arrestin activity by (i) ensuring an adequate supply for rapid quenching of the visual signal and (ii) limiting the availability of active monomeric species, thereby preventing inappropriate signal termination.

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors.

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

Pyrido[2,3-d]pyrimidin-5-ones: A Novel Class of Antiinflammatory Macrophage Colony-Stimulating Factor-1 Receptor Inhibitors†

A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.

Optimization of a Potent Class of Arylamide Colony-Stimulating Factor-1 Receptor Inhibitors Leading to Anti-inflammatory Clinical Candidate 4-Cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamide (JNJ-28312141).

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

Protein Engineering of the Colony-stimulating Factor-1 Receptor Kinase Domain for Structural Studies

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.