Role of Molecular Chaperones in G Protein ��5/Regulator of G Protein Signaling Dimer Assembly and G Protein �¦� Dimer Specificity

The G protein βγ subunit dimer (Gβγ) and the Gβ5/regulator of G protein signaling (RGS) dimer play fundamental roles in propagating and regulating G protein pathways, respectively. How these complexes form dimers when the individual subunits are unstable is a question that has remained unaddressed for many years. In the case of Gβγ, recent studies have shown that phosducin-like protein 1 (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, it is not known what fraction of the many Gβγ combinations is assembled this way or whether chaperones influence the specificity of Gβγ dimer formation. Moreover, the mechanism of Gβ5-RGS assembly has yet to be assessed experimentally. The current study was undertaken to directly address these issues. The data show that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1–4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results also show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way.Eukaryotic cells utilize receptors coupled to heterotrimeric GTP-binding proteins (G proteins)³ to mediate a vast array of responses ranging from nutrient-induced migration of single-celled organisms to neurotransmitter-regulated neuronal activity in the human brain (1). Ligand binding to a G protein-coupled receptor (GPCR) initiates GTP exchange on the G protein heterotrimer (composed of Gα, Gβ, and Gγ subunits), which in turn causes the release of Gα-GTP from the Gβγ dimer (2–4). Both Gα-GTP and Gβγ propagate and amplify the signal by interacting with effector enzymes and ion channels (1, 5). The duration and amplitude of the signal is dictated by receptor phosphorylation coupled with arrestin binding and internalization (6) and by regulators of G protein signaling (RGS) proteins, which serve as GTPase-activating proteins for the GTP-bound Gα subunit (7, 8). The G protein signaling cycle is reset as the inactive Gα-GDP reassembles with the Gβγ dimer and Gαβγ re-associates with the GPCR (5).To fulfill its essential role in signaling, the G protein heterotrimer must be assembled post-translationally from its nascent polypeptides. Significant progress has been made recently regarding the mechanism by which this process occurs. It has been clear for some time that the Gβγ dimer must assemble first, followed by subsequent association of Gα with Gβγ (9). What has not been clear was how Gβγ assembly would occur given the fact that neither Gβ nor Gγ is structurally stable without the other. An important breakthrough was the finding that phosducin-like protein 1 (PhLP1) functions as a co-chaperone with the chaperonin containing tailless complex polypeptide 1 (CCT) in the folding of nascent Gβ and its association with Gγ (10–15). CCT is an important chaperone that assists in the folding of actin and tubulin and many other cytosolic proteins, including many β propeller proteins like Gβ (16). PhLP1 has been known for some time to interact with Gβγ and was initially believed to inhibit Gβγ function (17). However, several recent studies have demonstrated that PhLP1 and CCT work together in a highly orchestrated manner to form the Gβγ dimer (10–15).Studies on the mechanism of PhLP1-mediated Gβγ assembly have focused on the most common dimer Gβ1γ2 (10, 13, 14), leaving open questions about the role of PhLP1 in the assembly of the other Gβγ combinations. These are important considerations given that humans possess 5 Gβ genes and 12 Gγ genes with some important splice variants (18, 19), resulting in more than 60 possible combinations of Gβγ dimers. Gβ1–4 share between 80 and 90% sequence identity and are broadly expressed (18, 19). Gβ5, the more atypical isoform, shares only ∼53% identity with Gβ1, carries a longer N-terminal domain, and is only expressed in the central nervous system and retina (20). The Gγ protein family is more heterogeneous than the Gβ family. The sequence identity of the 12 Gγ isoforms extends from 10 to 70% (21), and the Gγ family can be separated into 5 subfamilies (21–23). All Gγ proteins carry C-terminal isoprenyl modifications, which contribute to their association with the cell membrane, GPCRs, Gαs, and effectors (9). Subfamily I Gγ isoforms are post-translationally farnesylated, whereas all others are geranylgeranylated (22, 24).There is some inherent selectivity in the assembly of different Gβγ combinations, but in general Gβ1–4 can form dimers with most Gγ subunits (25). The physiological purpose of this large number of Gβγ combinations has intrigued researchers in the field for many years, and a large body of research indicates that GPCRs and effectors couple to a preferred subset of Gβγ combinations based somewhat on specific sequence complementarity, but even more so on cellular expression patterns, subcellular localization, and post-translational modifications (18).In contrast to Gβ1–4, Gβ5 does not interact with Gγ subunits in vivo, but it instead forms irreversible dimers with RGS proteins of the R7 family, which includes RGS proteins 6, 7, 9, and 11 (26). All R7 family proteins contain an N-terminal DEP (disheveled, Egl-10, pleckstrin) domain, a central Gγ-like (GGL) domain, and a C-terminal RGS domain (8, 26). The DEP domain interacts with the membrane anchoring/nuclear shuttling R7-binding protein, and the GGL domain binds to Gβ5 in a manner similar to other Gβγ associations (27, 28). Like Gβγs, Gβ5 and R7 RGS proteins form obligate dimers required for their mutual stability (26). Without their partner, Gβ5 and R7 RGS proteins are rapidly degraded in cells (26, 29). Gβ5-R7 RGS complexes act as important GTPase-accelerating proteins for G_i/oα and G_qα subunits in neuronal cells and some immune cells (26).It has been recently shown that all Gβ isoforms are able to interact with the CCT complex, but to varying degrees (15). Gβ4 and Gβ1 bind CCT better than Gβ2 and Gβ3, whereas Gβ5 binds CCT poorly (15). These results suggest that Gβ1 and Gβ4 might be more dependent on PhLP1 than the other Gβs, given the co-chaperone role of PhLP1 with CCT in Gβ1γ2 assembly. However, another report has indicated that Gγ2 assembly with Gβ1 and Gβ2 is more PhLP1-dependent than with Gβ3 and Gβ4 (30). Thus, it is not clear from current information whether PhLP1 and CCT participate in assembly of all Gβγ combinations or whether they contribute to the specificity of Gβγ dimer formation, nor is it clear whether they or other chaperones are involved in Gβ5-R7 RGS dimer formation. This report was designed to address these issues.     

Dimer Formation of a Stabilized Gβ1 Variant: A Structural and Energetic Analysis

In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol^− 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β strands 2 and 2′ and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120° rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gβ1 variants.     

The Cyclic Dimer of 5-Fluoro-2′-Deoxyuridylic Acid: A Potent Anticancer Agent

Abstract

As part of a synthetic program on cyclic oligomers of DNA, the cyclic dimer of 5-fluoro-2′-deoxyuridylic acid (FdUMP) was synthesized. The fully protected dimer 5 was obtained following Catlin and Cramer′s phosphotri-ester strategy. Autocondensation and deprotection then afforded the title compound 9 [cyclo(5FdUp5FdUp)] in excellent yield. In vitro, 9 proved slightly less active than FdUrd in inhibiting the proliferation of various murine and human tumor cells, but, in vivo, 9 was equally effective, and less toxic than 5-FdUrd in inhibiting adenocarcinoma tumor growth in mice.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

Understanding the Basis of Drug Resistance of the Mutants of αβ-Tubulin Dimer via Molecular Dynamics Simulations

The vital role of tubulin dimer in cell division makes it an attractive drug target. Drugs that target tubulin showed significant clinical success in treating various cancers. However, the efficacy of these drugs is attenuated by the emergence of tubulin mutants that are unsusceptible to several classes of tubulin binding drugs. The molecular basis of drug resistance of the tubulin mutants is yet to be unraveled. Here, we employ molecular dynamics simulations, protein-ligand docking, and MMPB(GB)SA analyses to examine the binding of anticancer drugs, taxol and epothilone to the reported point mutants of tubulin - T274I, R282Q, and Q292E. Results suggest that the mutations significantly alter the tubulin structure and dynamics, thereby weaken the interactions and binding of the drugs, primarily by modifying the M loop conformation and enlarging the pocket volume. Interestingly, these mutations also affect the tubulin distal sites that are associated with microtubule building processes.     

Utilization of a Cyclic Dimer and Linear Oligomers of ε-Aminocaproic Acid by Achrornobacter guttatus KI 72

A microorganism, strain KI 72 capable of utilizing ε-aminocaproic acid cyclic dimer as sole carbon and nitrogen sources was isolated from sludge and identified as Achromobacter guttatus. This bacteria utilized 1% of the cyclic dimer in a day and was not inhibited by the higher concentration of the dimer. The growth rate was independent of the cyclic dimer concentration in the medium, but the maximum cell concentration increased with the increase of substrate concentration. The cell yield was 0.7 mg dry cell/mg ε-aminocaproic acid cyclic dimer. Bacterial growth with the cyclic dimer as substrate was significantly stimulated by the addition of yeast extract. Ferric chloride was also stimulatory. Maximal growth was obtained in cultures incubated at pH 6 and at 33°C. Synthesized nylon oligomers, ranging from ε-aminocaproic acid up to its linear hexamer, were found to be catabolized by this organism.     

Characterization of Inhibitor-Bound α-Synuclein Dimer: Role of α-Synuclein N-Terminal Region in Dimerization and Inhibitor Binding

α-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other α-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit α-synuclein assembly with low micromolar IC₅₀ values, and that SDS-stable, noncytotoxic soluble α-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound α-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound α-synucleins in detail, we have prepared α-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated α-synuclein monomer and dimer were oxidized at all four methionine residues of α-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of α-synuclein. Ultra-high-field NMR analysis of inhibitor-bound α-synuclein dimers showed that the signals derived from the N-terminal region of α-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric α-synuclein. We propose that the N-terminal region of α-synuclein plays a key role in the formation of α-synuclein assemblies.     

A New Metabolite, 6-Oxo-7-azatridecanedioic Acid,a Microbial Product from Cyclic Dimer of ε-Caprolactam

A series of partially and heterogeneously N-acylated chitosans was prepared and isolated in 50 ~ 100% yields. The structure of N-acyl groups influenced the gelation. The minimum requirement for the gelation was defined as ca. 0.4 N-lauroyl (C₁₂), ca. 0.6 N-fatty acyl (C₃–C₁₀) or ca. 0.7 N-benzoyl groups per hexosaminide residue. However, the gelation did not occur with N-high fatty acyl (C₁₄-C₁₈) groups.1)     

Synthesis of a Carboxamide Linked T∗T Dimer with an Acyclic Nucleoside Unit and Its Incorporation in Oligodeoxynucleotides

Abstract

A T?T dimer with ? representing a 2′-OCH₂CH₂NHC(O)-4′ linkage connecting two nucleoside units was prepared by condensation of (S)-1-[2-(2-aminoethoxy)-3-(4,4′-dimethoxytrityloxy)propyl]thymine with 1,2-dideoxy-1-thyminyl-β-D-erythro-pento-furanuronic acid. The T?T dimer was incorporated in oligodeoxynucleotides and investigated for hybridization to DNA.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for?the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

β-Lactoglobulin's Conformational Requirements for Ligand Binding at the Calyx and the Dimer Interphase: a Flexible Docking Study

β-lactoglobulin (BLG) is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i) 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii) Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii) Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv) lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLG''s binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control binding.     

Structural and Mechanical Hierarchies in the α-Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.     

The Structure and Stability of the Disulfide-Linked γS-Crystallin Dimer Provide Insight into Oxidation Products Associated with Lens Cataract Formation

The reducing environment in the eye lens diminishes with age, leading to significant oxidative stress. Oxidation of lens crystallin proteins is the major contributor to their destabilization and deleterious aggregation that scatters visible light, obscures vision, and ultimately leads to cataract. However, the molecular basis for oxidation-induced aggregation is unknown. Using X-ray crystallography and small-angle X-ray scattering, we describe the structure of a disulfide-linked dimer of human γS-crystallin that was obtained via oxidation of C24. The γS-crystallin dimer is stable at glutathione concentrations comparable to those in aged and cataractous lenses. Moreover, dimerization of γS-crystallin significantly increases the protein’s propensity to form large insoluble aggregates owing to non-cooperative domain unfolding, as is observed in crystallin variants associated with early-onset cataract. These findings provide insight into how oxidative modification of crystallins contributes to cataract and imply that early-onset and age-related forms of the disease share comparable development pathways.     

The Synthesis of the Sixteen Possible 2′-O-Methyl MMI Dimer Phosphoramidites: Building Blocks for the Synthesis of Novel Antisense Oligonucleotides

Abstract

The synthesis of Methylene(methylimino) or MMI linked nucleoside dimers in all sixteen possible configurations has been accomplished via a reductive coupling of a nucleosidic aldehyde with an hydroxylamine. This has allowed us to prepare all of the necessary 2′-O-methyl MMI dimer building blocks necessary for use in an antisense motif.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the three N. crassa Gα subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3^Q208L allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.     

The Structure of Irisin Reveals a Novel Intersubunit β-Sheet Fibronectin Type III (FNIII) Dimer: IMPLICATIONS FOR RECEPTOR ACTIVATION*

Irisin was recently identified as a putative myokine that is induced by exercise. Studies suggest that it is produced by cleavage of the FNDC5 (fibronectin domain-containing protein 5) receptor; irisin corresponds to the extracellular receptor ectodomain. Data suggesting that irisin stimulates white-to-brown fat conversion have led to the hypothesis that it does so by binding an unknown receptor, thus functioning as a myokine. As brown fat promotes energy dissipation, myokines that elicit the transformation of white to brown fat have potentially profound benefits in the treatment of obesity and metabolic disorders. Understanding the molecular basis for such exercise-induced phenomena is thus of considerable interest. Moreover, FNDC5-like receptors are highly conserved and have been shown to be critical for neuronal development. However, the structural and molecular mechanisms utilized by these proteins are currently unknown. Here, we describe the crystal structure and biochemical characterization of the FNDC5 ectodomain, corresponding to the irisin myokine. The 2.28 Å structure shows that irisin consists of an N-terminal fibronectin III (FNIII)-like domain attached to a flexible C-terminal tail. Strikingly, the FNIII-like domain forms a continuous intersubunit β-sheet dimer, previously unobserved for any FNIII protein. Biochemical data confirm that irisin is a dimer and that dimerization is unaffected by glycosylation. This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors.     

Synergistic Binding of the Phosphorylated S233- and S259-Binding Sites of C-RAF to One 14-3-3ζ Dimer

C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen‐activated protein kinase/extracellular signal‐regulated kinase)-ERK (extracellular signal‐regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single‐site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single‐site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins.