The arginine finger of the Bloom syndrome protein: its structural organization and its role in energy coupling

RecQ family helicases are essential in maintaining chromosomal DNA stability and integrity. Despite extensive studies, the mechanisms of these enzymes are still poorly understood. Crystal structures of many helicases reveal a highly conserved arginine residue located near the γ-phosphate of ATP. This residue is widely recognized as an arginine finger, and may sense ATP binding and hydrolysis, and transmit conformational changes. We investigated the existence and role of the arginine finger in the Bloom syndrome protein (BLM), a RecQ family helicase, in ATP hydrolysis and energy coupling. Our studies by combination of structural modelling, site-directed mutagenesis and biochemical and biophysical approaches, demonstrate that mutations of residues interacting with the γ-phosphate of ATP or surrounding the ATP-binding sites result in severe impairment in the ATPase activity of BLM. These mutations also impair BLM's DNA-unwinding activities, but do not affect its ATP and DNA-binding abilities. These data allow us to identify R982 as the residue that functions as a BLM arginine finger. Our findings further indicate how the arginine finger is precisely positioned by the conserved motifs with respect to the γ-phosphate.     

A Conserved Negative Regulatory Region in αPAK: Inhibition of PAK Kinases Reveals Their Morphological Roles Downstream of Cdc42 and Rac1

αPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129–1143, 1997) resembling those of Cdc42^G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in αPAK). αPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42^G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding αPAK^83-149 potently inhibited GTPγS-bound Cdc42-mediated kinase activation of both αPAK and βPAK. Coexpression of this PAK inhibitor with Cdc42^G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1^G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of αPAK^83-149 completely blocked the phenotypic effects of hyperactive αPAK^L107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.     

Mapping the Structural and Dynamical Features of Kinesin Motor Domains

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.     

A model for how Gβγ couples Gα to GPCR

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine–proline–phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR–G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ–rhodopsin interactions may facilitate GPCR dimer transactivation.     

Substantial Conformational Change Mediated by Charge-Triad Residues of the Death Effector Domain in Protein-Protein Interactions

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.     

Common determinants in DNA melting and helicase-catalysed DNA unwinding by papillomavirus replication protein E1

E1 and T-antigen of the tumour viruses bovine papillomavirus (BPV-1) and Simian virus 40 (SV40) are the initiator proteins that recognize and melt their respective origins of replication in the initial phase of DNA replication. These proteins then assemble into processive hexameric helicases upon the single-stranded DNA that they create. In T-antigen, a characteristic loop and hairpin structure (the pre-sensor 1β hairpin, PS1βH) project into a central cavity generated by protein hexamerization. This channel undergoes large ATP-dependent conformational changes, and the loop/PS1βH is proposed to form a DNA binding site critical for helicase activity. Here, we show that conserved residues in BPV E1 that probably form a similar loop/hairpin structure are required for helicase activity and also origin (ori) DNA melting. We propose that DNA melting requires the cooperation of the E1 helicase domain (E1HD) and the origin binding domain (OBD) tethered to DNA. One possible mechanism is that with the DNA locked in the loop/PS1βH DNA binding site, ATP-dependent conformational changes draw the DNA inwards in a twisting motion to promote unwinding.     

The Structure of NtdA,a Sugar Aminotransferase Involved in the Kanosamine Biosynthetic Pathway in Bacillus subtilis,Reveals a New Subclass of Aminotransferases

NtdA from Bacillus subtilis is a sugar aminotransferase that catalyzes the pyridoxal phosphate-dependent equatorial transamination of 3-oxo-α-d-glucose 6-phosphate to form α-d-kanosamine 6-phosphate. The crystal structure of NtdA shows that NtdA shares the common aspartate aminotransferase fold (Type 1) with residues from both monomers forming the active site. The crystal structures of NtdA alone, co-crystallized with the product α-d-kanosamine 6-phosphate, and incubated with the amine donor glutamate reveal three key structures in the mechanistic pathway of NtdA. The structure of NtdA alone reveals the internal aldimine form of NtdA with the cofactor pyridoxal phosphate covalently attached to Lys-247. The addition of glutamate results in formation of pyridoxamine phosphate. Co-crystallization with kanosamine 6-phosphate results in the formation of the external aldimine. Only α-d-kanosamine 6-phosphate is observed in the active site of NtdA, not the β-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VI_β family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.     

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

Ligand Binding to the AMP-activated Protein Kinase Active Site Mediates Protection of the Activation Loop from Dephosphorylation

The AMP-activated protein kinase (AMPK) is a conserved signaling molecule in a pathway that maintains adenosine triphosphate homeostasis. Recent studies have suggested that low energy adenylate ligands bound to one or more sites in the γ subunit of AMPK promote the formation of an active, phosphatase-resistant conformation. We propose an alternative model in which the kinase domain association with the heterotrimer core results in activation of the kinase catalytic activity, whereas low energy adenylate ligands bound in the kinase active site promote phosphatase resistance. Purified Snf1 α subunit with a conservative, single amino acid substitution in the kinase domain is protected from dephosphorylation by adenosine diphosphate in the complete absence of the β and γ subunits. Staurosporine, a compound known to bind to the active site of many protein kinases, mediates strong protection from dephosphorylation to yeast and mammalian AMPK enzymes. The analog-sensitive Snf1-I132G protein but not wild type Snf1 exhibits protection from dephosphorylation when bound by the adenosine analog 2NM-PP1 in vitro and in vivo. These data demonstrate that ligand binding to the Snf1 active site can mediate phosphatase resistance. Finally, Snf1 kinase with an amino acid substitution at the interface of the kinase domain and the heterotrimer core exhibits normal regulation of phosphorylation in vivo but greatly reduced Snf1 kinase activity, supporting a model in which kinase domain association with the heterotrimer core is needed for kinase activation.     

Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.     

CLC Anion Channel Regulatory Phosphorylation and Conserved Signal Transduction Domains

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.     

NAR Breakthrough Article: Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N⁶-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH5_66–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.     

Hypothesis: bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine

The prokaryotic DNA polymerase III clamp loader complex loads the β clamp onto DNA to link the replication complex to DNA during processive synthesis and unloads it again once synthesis is complete. This minimal complex consists of one δ, one δ′ and three γ subunits, all of which possess an AAA+ module—though only the γ subunit exhibits ATPase activity. Here clues to underlying clamp loader mechanisms are obtained through Bayesian inference of various categories of selective constraints imposed on the γ and δ′ subunits. It is proposed that a conserved histidine is ionized via electron transfer involving structurally adjacent residues within the sensor 1 region of γ's AAA+ module. The resultant positive charge on this histidine inhibits ATPase activity by drawing the negatively charged catalytic base away from the active site. It is also proposed that this arrangement is disrupted upon interaction of DNA with basic residues in γ implicated previously in DNA binding, regarding which a lysine that is near the sensor 1 region and that is highly conserved both in bacterial and in eukaryotic clamp loader ATPases appears to play a critical role. γ ATPases also appear to utilize a trans-acting threonine that is donated by helix 6 of an adjacent γ or δ′ subunit and that assists in the activation of a water molecule for nucleophilic attack on the γ phosphorous atom of ATP. As eukaryotic and archaeal clamp loaders lack most of these key residues, it appears that eubacteria utilize a fundamentally different mechanism for clamp loader activation than do these other organisms.     

Constitutively Oxidized CXXC Motifs within the CD3 Heterodimeric Ectodomains of the T Cell Receptor Complex Enforce the Conformation of Juxtaposed Segments

The CD3ϵγ and CD3ϵδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αβ heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ϵ, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction.     

Phosphorylation of C3a Receptor at Multiple Sites Mediates Desensitization, β-Arrestin-2 Recruitment and Inhibition of NF-κB Activity in Mast Cells

Background

Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined.

Methodology/Principal Findings

We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58±3.8% decrease in agonist-induced C3aR phosphorylation there was no change in β-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40±1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74±2.4% decreases in β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca²⁺ mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%.

Conclusion/Significance

This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways.     

Contribution of each Trp residue toward the intrinsic fluorescence of the Giα1 protein

G_iα1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of G_iα1 bound to GDP•AMF or GTPγS with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the α-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP•AMF or GTPγS, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTPγS, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a π-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic π-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of G_iα1.