Cooperative non-specific DNA binding of the N-terminal core of the cyclic AMP receptor protein of Escherichia coli and its modulation by cyclic AMP

The non-specific DNA binding of CRP and its N-terminal core, alpha CRP, to a 298 base pair DNA fragment, in the presence and absence of cAMP, has been studied using the nitrocellulose filter binding technique and analysed quantitatively using the theory of Clore et al. [J. Mol. Biol. (1982) 155, 447-466]. It is shown that both CRP and alpha CRP bind cooperatively to DNA. At an ionic strength of 100 mM and pH 7.5, the intrinsic equilibrium association constant for the binding of alpha CRP to DNA is approximately 10-times smaller than that for CRP, but the cooperativity parameter is approximately 17-times larger for alpha CRP than CRP. cAMP exerts its effect solely on the intrinsic equilibrium constant and does not alter the cooperativity. In the case of alpha CRP, cAMP reduces the intrinsic equilibrium association constant by a factor of 3, in contrast to the case of CRP where cAMP increases it by a factor of 3. The possible location of the DNA binding site present in the N-terminal core of CRP is discussed in the light of crystallographic data on the cAMP . CRP complex [McKay et al. (1982) J. Biol. Chem. 257, 9518-9524].     

Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli

Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

The role of loops 3 and 4 in the interdomains and intersubunits communication of E. coli cAMP receptor protein

Cyclic AMP serves as an intracellular messenger in cells and regulates a variety of biological functions by transmitting information through proteins. These proteins of different functions all consist of a cAMP-binding motif, and the structure of this motif is highly conserved with an exception of the loop 3 and 4. In current study, cAMP receptor protein was employed as a model system to investigate the function of the two loops. The results indicated that the loop 3 involves in the intersubunits communication of CRP, whereas the loop 4 involves in cAMP binding and interdomains communication.     

Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon

The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose. Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the -35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at -92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the alpha subunit of RNA polymerase deleted for the entire C-terminal domain (alpha-Delta235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the alpha subunit (alpha-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on alpha-CTD. Overall, our results suggest that CRP interaction with alpha-CTD may not be required for rhaBAD activation; however, alpha-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

Roles of intraloops-2 and -3 and the proximal C-terminus in signalling pathway selection from the human calcium-sensing receptor

The calcium-sensing receptor (CaSR) couples to signalling pathways via intracellular loops 2 and 3, and the C-terminus. However, the requirements for signalling are largely undefined. We investigated the impacts of selected point mutations in iL-2 (F706A) and iL-3 (L797A and E803A), and a truncation of the C-terminus (R866X) on extracellular Ca²⁺ (Ca²⁺_o)-stimulated phosphatidylinositol-specific phospholipase-C (PI-PLC) and various other signalling responses. CaSR-mediated activation of PI-PLC was markedly attenuated in all four mutants and similar suppressions were observed for Ca²⁺_o-stimulated ERK_1/2 phosphorylation. Ca²⁺_o-stimulated intracellular Ca²⁺ (Ca²⁺_i) mobilization, however, was relatively preserved for the iL-2 and iL-3 mutants and suppression of adenylyl cyclase was unaffected by either E803A or R866X. The CaSR selects for specific signalling pathways via the proximal C-terminus and key residues in iL-2, iL-3.     

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1)

Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.     

Inactivation of lipoprotein lipase in 3T3-L1 adipocytes by angiopoietin-like protein 4 requires that both proteins have reached the cell surface

Lipoprotein lipase (LPL) and angiopoietin-like protein 4 (Angptl4) were studied in 3T3-L1 adipocytes. Transfections of the adipocytes with Angptl4 esiRNA caused reduction of the expression of Angptl4 to about one fourth of that in cells treated with vehicle only. This resulted in higher levels of LPL activity both on cell surfaces (heparin-releasable) and in the medium, while LPL activity within the cells remained unaffected. This demonstrated that even though both proteins are made in the same cell, Angptl4 does not inactivate LPL during intracellular transport. Most of the Angptl4 protein was present as covalent dimers and tetramers on cell surfaces, while within the cells there were only monomers. LPL gradually lost activity when incubated in medium, but there was no marked difference between conditioned medium from normal cells (rich in Angptl4) and medium after knockdown of Angptl4. Hence Angptl4 did not markedly accelerate inactivation of LPL in the medium. Experiments with combinations of different cells and media indicated that inactivation of LPL occurred on the surfaces of cells producing Angptl4.     

The mechanism of sugar-mediated catabolite repression of the propionate catabolic genes in Escherichia coli

Carbon catabolite repression (CCR) is a well-known phenomenon that involves the preferential utilization of glucose as a carbon source. Cyclic adenosine monophosphate (cAMP) and the cAMP receptor protein (CRP) mediate CCR. Recently, a second CCR hierarchy that leads to the preferential consumption of arabinose over xylose, mediated by arabinose-bound AraC, has been identified. In this study, we report yet another CCR hierarchy that causes the preferential utilization of sugars (arabinose, galactose, glucose, mannose, and xylose) over a short-chain fatty acid (propionate). Expression of the propionate catabolic (prpBCDE) genes is down-regulated in the presence of these sugars. Sugar-mediated repression of the propionate catabolic genes is independent of sugar-specific regulators such as AraC and dependent on global regulators of sugar transport such as the cAMP-CRP complex and the Phosphotransferase System (PTS). Inhibition of the prpBCDE promoter is encountered during rapid sugar uptake and metabolism. This unique regulatory crosstalk between sugar metabolism and fatty acid metabolism may help provide new insights into CRP-dependent catabolite repression acting in conjunction with non-carbohydrate metabolism.     

The structure and unusual protein chemistry of hypoxic response protein 1, a latency antigen and highly expressed member of the DosR regulon in Mycobacterium tuberculosis

Mycobacterium tuberculosis adapts to cellular stresses such as decreased oxygen concentration, at least in part, by upregulation of the dormancy survival regulon, which is thought to be important for the bacterium's ability to enter a persistent state in its human host. We have determined the structure of hypoxic response protein 1, a protein encoded by one of the most strongly upregulated genes in the dormancy survival regulon. Hypoxic response protein 1 is an example of a ‘cystathionine-β-synthase-domain-only’ protein; however, unlike other cystathionine-β-synthase domains, it does not appear to bind AMP. The protein is proteolytically sensitive at its C-terminus and contains two unexpected disulfide bonds, one of which appears resistant to reducing agents in solution and is, therefore, most likely buried in the protein and is not solvent-accessible. We show that the protein is secreted from the bacterium in hypoxic in vitro culture and does not accumulate in the bacterial cell wall. The biological function of the protein remains unclear, but we suggest that it may contribute to the modulation of the host immune response. The work reported advances our understanding of the chemistry and cell biology of this intriguing and potentially important protein, and establishes a structural framework for future functional and immunological studies.     

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.     

Characterization of the interaction between lysyl-tRNA synthetase and laminin receptor by NMR

Lysyl-tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin-induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)-based study, we show that the anticodon-binding domain of KRS binds directly to the C-terminal region of 37LRP, and the previously found inhibitors BC-K-01 and BC-K-YH16899 interfere with KRS–37LRP binding. In addition, the anticodon-binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS–LR interaction on the cell surface.     

Chemical composition, intake by sheep, and in situ disappearance in cannulated cows of bermudagrass hayed at two moisture concentrations and treated with a non-viable Lactobacillus-lactic acid preservative

Bermudagrass [Cynodon dactylon (L.) Pers.] is commonly used for grazing and haying in the southern USA, but hay curing can be challenging due to frequent rainfall events during spring and early summer. An existing stand of ‘Greenfield’ bermudagrass was divided into 12 plots using a randomized complete block design with a 2×2 factorial treatment arrangement to evaluate the influence of a non-viable Lactobacillus-lactic acid preservative and moisture concentration at baling on chemical composition, intake by sheep, and in situ disappearance in cattle. At time of mowing, half of the plots in each block were either spray-treated (T) or not treated (U) with 81 mL/t forage dry matter (DM) of the preservative solution. Hay was then baled at target moisture concentrations of either 174 g/kg DM (L) or 267 g/kg DM (H). Maximum temperature and heating degree days were greater (P<0.05) from H compared with L during the 42-d storage period. An interaction between spray and moisture treatments tended (P<0.10) to affect recovery of DM; recoveries for LT (0.992) differed (P<0.10) from HT (0.913), but LU and HU were intermediate between the spray-treated hays, and did not differ from either (P>0.10). Post-storage nutritive value was largely influenced by moisture treatments only. Intake and digestibility, and in situ DM disappearance of these same hays were determined using 16 wether lambs (43 ± 3.7 kg initial BW), or six ruminally cannulated cows (617 ± 3.5 kg initial BW), respectively. Dry matter intake by sheep was not affected by either treatment factor (P>0.05), but DM digestibility and digestible DM intake were greater (P<0.05) from U compared with T. The in situ immediately soluble DM portion was greater from (P<0.05) L compared with H, but the reverse was true for the potentially degradable DM fraction. The lag time tended (P<0.10) to be greater from H compared with L. Treating bermudagrass with a non-viable Lactobacillus acidophilus-lactic acid spray product at time of baling may not offset the negative effects on forage quality and digestibility of baling bermudagrass hay at excessive moisture concentrations.