Leslie E. Eisele

Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.

Mycobacterium tuberculosis Rv3586 (DacA) Is a Diadenylate Cyclase That Converts ATP or ADP into c-di-AMP

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacAs enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+ or Co2+. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacAs diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.

The Discovery of a Novel R-phycoerythrin from an Antarctic Red Alga

A novel biliprotein, named R-phycoerythrin IV, has been discovered. It absorbs blue light better than any other known red algal biliprotein. The protein was found in Phyllophora antarctica, a benthic macroalga, which grows beneath the coastal waters of McMurdo Sound, Antarctica. Fluorescence emission and fluorescence excitation polarization spectroscopy demonstrated that R-phycoerythrin IV behaved as a typical R-phycoerythrin in the functioning of energy migration and has an emission maximum at 577 nm. The circular dichroism (CD) spectrum of the chromophores was compared with visible absorption spectrum, and both were deconvoluted. This process showed the energy states of various individual chromophores. The molecular weight of the protein suggested a α6β6γ polypeptide structure, and far UV CD studies revealed polypeptides with highly α-helical secondary structures. Dynamic light scattering indicated that the protein had a 5.54 nm radius, and its shape was nonspherical. R-phycoerythrin was also purified from a second benthic Antarctic red alga, Iridaea cordata. Its spectroscopic properties were similar to those of some R-phycoerythrins from nonpolar regions. The unique spectroscopic properties of R-phycocerythrin IV may help enable the alga to occupy its niche deeper in the water column than the red alga that has the typical R-phycoerythrin.

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from α-fetoprotein

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.

Zinc induces dimerization of the class II major histocompatibility complex molecule that leads to cooperative binding to a superantigen.

Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vβ elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306–318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn2+ is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn2+. However, in the presence of Zn2+, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn2+-induced DR1 dimer. In the presence of Zn2+, cooperative binding of MAM to the DR1 dimer was also observed.

Some physical properties of an unusual C-phycocyanin isolated from a photosynthetic thermophile

Abstract A cyanobacterium, which is a photosynthetic thermophile, has been grown in the laboratory at 66–70°C. This organism, Synechococcus lividus (SyI), was isolated from a hot spring in Yellowstone National Park where it grows at 68–73°C. The biliprotein, C-phycocyanin, has been purified, and some of its physical properties studied. When carefully prepared, the protein is obtained in a homogeneous form at about 600000 molecular weight. Its visible absorption spectrum has a maximum at 608 nm, which is blue shifted to a higher energy maximum than any other known C-phycocyanin. Studies are presented showing that the unique spectrum is not a function of protein aggregation, chemically changed chromophores, number of chromophores, linker polypeptides, temperature of measurement, or of its thermophilic origin. To produce this spectrum, one or more of the chromophores must be affected by apoprotein differently that it is affected in other proteins. The circular dichroism spectra of the protein have been studied, and the aggregation was monitored by fluorescence polarization and gel-filtration column chromatography. A method to prepare SyI monomers was developed. Rods, monomers, trimers, and hexamers were all shown to have blue-shifted absorption maxima. Three other C-phycocyanins — one from a mesophilic and two thermophilic cyanobacteria — were compared with the S. lividus (SyI) protein, which was found to be the most thermally stable and most resistent to dissociation.

Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat

C-Phycocyanin, a biliprotein, was purified from the red alga, Cyanidium caldarium. This alga grows at temperatures up to 57 degrees C, a very high temperature for a eukaryote, and at pH values down to 0.05. Using the chromophores on C-phycocyanin as naturally occurring reporter groups, the effects of temperature on the stability of the protein were studied by circular dichroism and absorption spectroscopy. The protein was unchanged from 10 to 50 degrees C, which indicates that higher temperatures are not required to cause the protein to be photosynthetically active. At 60 and 65 degrees C, which are above the temperatures at which the alga can survive, the protein undergoes irreversible denaturation. Gel-filtration column chromatography demonstrated that the irreversibility is caused by the dissociation of the trimeric protein to its constitutive polypeptides. Upon cooling, the alpha and beta polypeptides did not reassemble to the trimer. Unlike phycocyanins 645 and 612, the C-phycocyanin does not show a reversible conformational change at moderately high temperatures. At constant temperature, the C-phycocyanin was more stable than a mesophilic counterpart. It is designated a temperature-resistant protein.

Studies on R-phycoerythrins from two Antarctic marine red algae and a mesophilic red alga

Abstract R-phycoerythrin was purified from two benthic red algae, Iridaea cordata and Phyllophora antarctica, obtained growing at −2°C under thick sea ice off the coast of Antarctica. For the I. cordata protein, the molecular mass was 245,000 Da, and its secondary structure was 60% α helix, 17% β sheet, 16% turn, and 7% other. The light-harvesting faculties of the I. cordata protein resembled those of R-phycoerythrins from mesophilic red algae and were distinctive from the novel R-phycoerythrin from P. antarctica. Deconvolution of the visible absorption spectrum of R-phycoerythrin from I. cordata indicated a minimum of five component bands having maxima at 568, 558, 534, 496, and 481 nm. R-phycoerythrins from the mesophilic Porphyra tenera and psychrophilic Phyllophora antarctica had the same five bands. The protein from Phyllophora antarctica obtained its unique spectrum from a more intense component at 482 nm, and a less intense band at 533 nm. This change was probably produced by a replacement of phycoerythrobilin by phycourobilin. A temperature study of the circular dichroism CD was obtained for R-phycoerythrin from I. cordata from 4 to 80°C. Laser time-resolved fluorescence studies on R-phycoerythrin showed bilin to bilin energy transfer with a 60.2-ps lifetime, which should occur by the Förster resonance. The similarities in spectra between the proteins from I. cordata and Porphyra tenera and the different spectrum for the protein from Phyllophora antarctica show that only particular antarctic habitats require unique R-phycoerythrins.

Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific for Ricin Toxin’s Binding Subunit (RTB)

Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or “nanobody”) specific for ricin’s enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1∶10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Crystal structures of T cell receptor β chains related to rheumatoid arthritis

The crystal structures of the Vβ17+ β chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5–1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5–1F104→Y/C187→S) forms, respectively. These TCR β chains form homo‐dimers in solution and in crystals. Structural comparison reveals that the main‐chain conformations in the CDR regions of the C5–1 and SF4 Vβ17 closely resemble those of a Vβ17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three Vβ17 structures. At the side‐chain level, conformational differences were observed at the CDR2 regions between our two ligand‐free forms and the bound JM22 form. Other significant differences were observed at the Vβ regions 8–12, 40–44, and 82–88 between C5–1/SF4 and JM22 Vβ17, implying that there is considerable variability in the structures of very similar β chains. Structural alignments also reveal a considerable variation in the Vβ–Cβ associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the Vβ domains of known TCR structures indicate that there are significant similarities among Vβ regions that are MAM‐reactive, whereas there appear to be significant structural differences among those Vβ regions that lack MAM‐reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.