Variable CGRP family peptide signaling durations and the structural determinants thereof.

Calcitonin gene-related peptides alpha and beta (αCGRP, ßCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, -2, or -3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1-47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8-47) and AM2/IMD-53 exhibited even longer duration signaling than the 1-47 fragment. AM2/IMD(8-47) was a striking 8-fold longer acting than AM(13-52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1-52) and (13-52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. ßCGRP was âˆ¼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of ßCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

Time- and cost-efficient bacterial expression and purification of potato apyrase.

Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of ∼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.

Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization.

The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.

Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.

Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a ß-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

Selective CGRP and adrenomedullin peptide binding by tethered RAMP-calcitonin receptor-like receptor extracellular domain fusion proteins.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.

Reconstitution of R-spondin:LGR4:ZNRF3 adult stem cell growth factor signaling complexes with recombinant proteins produced in Escherichia coli.

R-Spondins are secreted glycoproteins (RSPO1-RSPO4) that have proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine-rich repeat (LRR)-containing seven-transmembrane receptors LGR4-LGR6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present a methodology for the bacterial expression and purification of the signaling competent, cysteine-rich Fu1-Fu2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1-14, and the human ZNRF3 ECD. In a cell-based signaling assay, the nonglycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to those of mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nanomolar affinities that decreased in the following order in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: RSPO4 > RSPO2 > RSPO3 > RSPO1. RSPO-receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinities that decreased in the following order: RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and suggest that the stronger signaling potencies of RSPO2 and -3 result from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.

Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation.

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

Structural basis for parathyroid hormone-related protein binding to the parathyroid hormone receptor and design of conformation-selective peptides.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic alpha-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally "unwound." The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.

Molecular recognition of corticotropin-releasing factor by its G-protein-coupled receptor CRFR1.

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

Structure-function analysis of glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49) of Escherichia coli.

Glutamine synthetase adenylyltransferase (ATase) regulates the activity of glutamine synthetase by adenylylation and deadenylylation in response to signals of nitrogen and carbon status: glutamine, alpha-ketoglutarate, and the uridylylated and unmodified forms of the PII signal transduction protein. ATase consists of two conserved nucleotidyltransferase (NT) domains linked by a central region of approximately 200 amino acids. Here, we study the activities and regulation of mutated and truncated forms of ATase. Our results indicate the following. (i) The N-terminal NT domain contained the adenylyl-removing (AR) active site, and the C-terminal NT domain contained the adenylyltransferase (AT) active site. (ii) The enzyme contained a glutamine binding site, and glutamine increased the affinity for PII. (iii) The enzyme appeared to contain multiple sites for the binding of PII and PII-UMP. (iv) Truncated versions of ATase missing the C-terminal (NT) domain lacked both AT and AR activity, suggesting a role for the C-terminal NT domain in both activities. (v) The purified C-terminal NT domain and larger polypeptides containing this domain had significant basal AT activity, which was stimulated by glutamine. These polypeptides were indifferent to PII and PII-UMP, or their ATase activity was inhibited by either PII or PII-UMP. (vi) Certain point mutations in the central region or an internal deletion removing most of this part of the protein eliminated the AR activity and eliminated activation of the AT activity by PII, while not eliminating the binding of PII or PII-UMP. That is, these mutations in the central region appeared to destroy the communication between the PII and PII-UMP binding sites and the AT and AR active sites. (vii) Certain mutations in the central region of ATase appeared to dramatically improve the binding of glutamine to the enzyme. (viii) While the isolated AT and AR domains of ATase bound poorly to PII and PII-UMP, these domains bound PII and PII-UMP significantly better when linked to the central region of ATase. Together, our results indicate a highly coordinated enzyme, in which the AT and AR domains participate in each other's regulation and distant regulatory sites are in communication with each other. A model for the regulation of ATase by glutamine, PII, and PII-UMP consistent with all data is presented.

Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-L-homocysteine.

The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-L-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-L-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 A resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 A, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central alpha/beta-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.

Crystal structure of the C-terminal domain of the two-component system transmitter protein nitrogen regulator II (NRII; NtrB), regulator of nitrogen assimilation in Escherichia coli.

The kinase/phosphatase nitrogen regulator II (NRII, NtrB) is a member of the transmitter protein family of conserved two-component signal transduction systems. The kinase activity of NRII brings about the phosphorylation of the transcription factor nitrogen regulator I (NRI, NtrC), causing the activation of Ntr gene transcription. The phosphatase activity of NRII results in the inactivation of NRI-P. The activities of NRII are regulated by the signal transduction protein encoded by glnB, PII protein, which upon binding to NRII inhibits the kinase and activates the phosphatase activity. The C-terminal ATP-binding domain of NRII is required for both the kinase and phosphatase activities and contains the PII binding site. Here, we present the crystal structure of the C-terminal domain of a mutant form of NRII, NRII-Y302N, at 1.6 A resolution and compare this structure to the analogous domains of other two-component system transmitter proteins. While the C-terminal domain of NRII shares the general tertiary structure seen in CheA, PhoQ, and EnvZ transmitter proteins, it contains a distinct beta-hairpin projection that is absent in these related proteins. This projection is near the site of a well-characterized mutation that reduces the binding of PII and near other less-characterized mutations that affect the phosphatase activity of NRII. Sequence alignment suggests that the beta-hairpin projection is present in NRII proteins from various organisms, and absent in other transmitter proteins from Escherichia coliK-12. This unique structural element in the NRII C-terminal domain may play a role in binding PII or in intramolecular signal transduction.