Triad Motif Research Articles

The alternate triad motif of the poly(ADP-ribose) polymerase-like domain of the human zinc finger antiviral protein is essential for its antiviral activity

The human zinc finger antiviral protein (hZAP) gene is spliced to yield a short (hZAP-S) and a long (hZAP-L) isoform. The long isoform possesses a poly(ADP-ribose) polymerase (PARP)-like domain in its C-terminus predicted to be inactive due to alterations in its triad motif compared with bona fide PARPs. Using Sindbis virus as prototype member of alphaviruses we confirmed that hZAP-L is a more potent inhibitor of alphaviruses than hZAP-S. Specific small interfering RNA knockdown of hZAP-L but not hZAP-S demonstrated a role of endogenous hZAP-L in restriction of alphavirus replication. Whilst single amino-acid substitutions in the triad motif of hZAP-L's PARP-like domain reduced the antiviral activity, exchange of all three triad motif residues to alanine or to the amino acids of active PARPs virtually abolished the antiviral effect. Contrary to previous assumptions, these results indicate an essential function of the PARP-like domain in hZAP-L's antiviral activity.

Tc1-like transposable elements in plant genomes.

BackgroundThe Tc1/mariner superfamily of transposable elements (TEs) is widespread in animal genomes. Mariner-like elements, which bear a DDD triad catalytic motif, have been identified in a wide range of flowering plant species. However, as the founding member of the superfamily, Tc1-like elements that bear a DD34E triad catalytic motif are only known to unikonts (animals, fungi, and Entamoeba).ResultsHere we report the identification of Tc1-like elements (TLEs) in plant genomes. These elements bear the four terminal nucleotides and the characteristic DD34E triad motif of Tc1 element. The two TLE families (PpTc1, PpTc2) identified in the moss (Physcomitrella patens) genome contain highly similar copies. Multiple copies of PpTc1 are actively transcribed and the transcripts encode intact full length transposase coding sequences. TLEs are also found in angiosperm genome sequence databases of rice (Oryza sativa), dwarf birch (Betula nana), cabbage (Brassica rapa), hemp (Cannabis sativa), barley (Hordium valgare), lettuce (Lactuta sativa), poplar (Populus trichocarpa), pear (Pyrus x bretschneideri), and wheat (Triticum urartu).ConclusionsThis study extends the occurrence of TLEs to the plant phylum. The elements in the moss genome have amplified recently and may still be capable of transposition. The TLEs are also present in angiosperm genomes, but apparently much less abundant than in moss.

Birth of Three Stowaway-like MITE Families via Microhomology-Mediated Miniaturization of a Tc1/Mariner Element in the Yellow Fever Mosquito

Eukaryotic genomes contain numerous DNA transposons that move by a cut-and-paste mechanism. The majority of these elements are self-insufficient and dependent on their autonomous relatives to transpose. Miniature inverted repeat transposable elements (MITEs) are often the most numerous nonautonomous DNA elements in a higher eukaryotic genome. Little is known about the origin of these MITE families as few of them are accompanied by their direct ancestral elements in a genome. Analyses of MITEs in the yellow fever mosquito identified its youngest MITE family, designated as Gnome, that contains at least 116 identical copies. Genome-wide search for direct ancestral autonomous elements of Gnome revealed an elusive single copy Tc1/Mariner-like element, named as Ozma, that encodes a transposase with a DD37E triad motif. Strikingly, Ozma also gave rise to two additional MITE families, designated as Elf and Goblin. These three MITE families were derived at different times during evolution and bear internal sequences originated from different regions of Ozma. Upon close inspection of the sequence junctions, the internal deletions during the formation of these three MITE families always occurred between two microhomologous sites (6–8 bp). These results suggest that multiple MITE families may originate from a single ancestral autonomous element, and formation of MITEs can be mediated by sequence microhomology. Ozma and its related MITEs are exceptional candidates for the long sought-after endogenous active transposon tool in genetic control of mosquitoes.

Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe(2+) to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7-1.98 Å resolution. The resting state structure displays an octahedral coordination for Fe(2+) with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe(2+), and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe(2+) coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O2-dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.

Molecular Modeling Comparison of the Performance of NS5b Polymerase Inhibitor (PSI-7977) on Prevalent HCV Genotypes

The current available treatment for hepatitis C virus (HCV)-the causative of liver cirrhosis and development of liver cancer-is a dual therapy using modified interferon and ribavirin. While this regimen increases the sustained viral response rate up to 40-80% in different genotypes, unfortunately, it is poorly tolerated by patients. PSI-7977, a prodrug for PSI-7409, is a Non-Structural 5b (NS5b) polymerase nucleoside inhibitor that is currently in phase III clinical trials. The activated PSI-7977 is a direct acting antiviral (DAA) drug that acts on NS5b polymerase of HCV through a coordination bond with the two Mg(+2) present at the GDD active site motif. The present work utilizes a molecular modeling approach for studying the interaction between the activated PSI-7977 and the 12 amino acids constituting a 5Å region surrounding the GDD active triad motif for HCV genotypes 1a, 2b, 3b and 4a. The analysis of the interaction parameters suggests that PSI-7977 is probably a better DAA drug for HCV genotypes 1a and 3b rather than genotypes 2b and 4a.

Emerging themes in radical SAM chemistry

Enzymes in the radical SAM (RS) superfamily catalyze a wide variety of reactions through unique radical chemistry. The characteristic markers of the superfamily include a [4Fe-4S] cluster coordinated to the protein via a cysteine triad motif, typically CX(3)CX(2)C, with the fourth iron coordinated by S-adenosylmethionine (SAM). The SAM serves as a precursor for a 5'-deoxyadenosyl radical, the central intermediate in nearly all RS enzymes studied to date. The SAM-bound [4Fe-4S] cluster is located within a partial or full triosephosphate isomerase (TIM) barrel where the radical chemistry occurs protected from the surroundings. In addition to the TIM barrel and a RS [4Fe-4S] cluster, many members of the superfamily contain additional domains and/or additional Fe-S clusters. Recently characterized superfamily members are providing new examples of the remarkable range of reactions that can be catalyzed, as well as new structural and mechanistic insights into these fascinating reactions.

Motifs insights from engineering systems architectures

“Network Motifs” is a research area in “Network Science” which has captured the attention of many researchers. Complex biological and social networks have displayed presence of some triad motifs far in excess (or short) of their expected values. Some of these over(under)-represented motifs have explained the basic functionality of systems, for example, in the sensory transcription networks of biology, over-represented motifs are shown to perform signal processing tasks. This suggests purposeful, selective retention of these motifs in the studied biological systems. Another interesting feature is the high correlation of triad motif significance profiles (MSPs) of all systems that belong to a family of naturally grouped systems, thereby suggesting that all the systems in a family have the same function to perform and hence the correlation. Engineering systems also display over(under)-represented motifs. The MSPs of a family of naturally grouped engineering systems show high correlation. Unlike biological and social networks, engineering systems are designed by humans and offer an opportunity for investigation based on known design rules. We show that over(under)-represented motifs in engineering systems are not purposefully retained/avoided to perform functions but are a natural consequence of design by decomposition. We also discover that naturally grouped systems have remarkably correlated in(out) degree distribution across nodes resulting in a high correlation in MSP. Therefore, we argue that the idea of network motifs has no significance in engineering systems (unlike biological and other evolutionary systems), and we caution the engineering research community to be careful while drawing conclusions based on network motifs. We report a remarkable correlation of in(out) degree distribution of systems within a family of engineering systems. We further show that biological and social networks also display signs of decomposition. They also show a high correlation of in–out degree distribution of nodes for systems within a family. This opens up an interesting opportunity to investigate these systems through their observed decomposition. © 2012 Wiley Periodicals, Inc. Complexity, 2012 © 2012 Wiley Periodicals, Inc.

Diverging Mechanisms of Activation of Chemokine Receptors Revealed by Novel Chemokine Agonists

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions.

Structural Insights into the Novel Diadenosine 5′,5‴- P1, P4-Tetraphosphate Phosphorylase from Mycobacterium tuberculosis H37Rv

Rv2613c is a diadenosine 5′,5‴- P 1, P 4-tetraphosphate (Ap 4A) phosphorylase from Mycobacterium tuberculosis H37Rv. Sequence analysis suggests that Rv2613c belongs to the histidine triad (HIT) motif superfamily, which includes HIT family diadenosine polyphosphate (Ap n A) hydrolases and Ap 4A phosphorylases. However, the amino acid sequence of Rv2613c is more similar to that of HIT family Ap n A hydrolases than to that of typical Ap 4A phosphorylases. Here, we report the crystal structure of Rv2613c, which is the first structure of a protein with Ap n A phosphorylase activity, and characterized the structural basis of its catalytic activity. Our results showed that the structure of Rv2613c is similar to those of other HIT superfamily proteins. However, Asn139, Gly146, and Ser147 in the active site of Rv2613c replace the corresponding Gln, Gln, and Thr residues that are normally found in HIT family Ap n A hydrolases. Furthermore, analyses of Rv2613c mutants revealed that Asn139, Gly146, and Ser147 are important active-site residues and that Asn139 has a critical role in catalysis. The position of Gly146 might influence the phosphorylase activity. In addition, the tetrameric structure of Rv2613c and the presence of Trp160 might be essential for the formation of the Ap 4A binding site. These structural insights into Rv2613c may facilitate the development of novel structure-based inhibitors for treating tuberculosis.

Olefin Cis-Dihydroxylation with Bio-Inspired Iron Catalysts. Evidence for an FeII/FeIV Catalytic Cycle

Iron(II) complexes of a series of N-acylated dipyridin-2-ylmethylamine ligands (R-DPAH) have been investigated as catalysts for the cis-dihydroxylation of olefins to model the action of Rieske dioxygenases that catalyze arene cis-dihydroxylation. The Rieske dioxygenases have a mononuclear iron active site coordinated to a 2-histidine-1-carboxylate facial triad motif. The R-DPAH ligands are designed to provide a facial N,N,O-ligand set that mimics the enzyme active site. The iron(II) complexes of the R-DPAH ligands activate H(2)O(2) to effect the oxidation of olefin substrates into cis-diol products. As much as 90% of the H(2)O(2) oxidant is converted into cis-diol, but a large excess of olefin is required to achieve the high conversion efficiency. Reactivity and mechanistic comparisons with the previously characterized Fe(TPA)/H(2)O(2) catalyst/oxidant combination (TPA = tris(pyridin-2-ylmethyl)amine) lead us to postulate an Fe(II)/Fe(IV) redox cycle for the Fe(R-DPAH) catalysts in which an Fe(IV)(OH)(2) oxidant carries out the cis-hydroxylation of olefins. This hypothesis is supported by three sets of observations: (a) the absence of a lag phase in the conversion of the H(2)O(2) oxidant into a cis-diol product, thereby excluding the prior oxidation of the Fe(II) catalyst to an Fe(III) derivative as established for the Fe(TPA) catalyst; (b) the incorporation of H(2)(18)O into the cis-diol product, thereby requiring O-O bond cleavage to occur prior to cis-diol formation; and (c) the formation of cis-diol as the major product of cyclohexene oxidation, rather than the epoxide or allylic alcohol products more commonly observed in metal-catalyzed oxidations of cyclohexene, implicating an oxidant less prone to oxo transfer or H-atom abstraction.

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.

Supramolecular Cobaloxime Assemblies for H2 Photocatalysis: An Initial Solution State Structure−Function Analysis

In this report, we have investigated the correlations between structure and light-induced electron transfer of one known and three new axially coordinated cobaloxime-based supramolecular photocatalysts for the reduction of protons to hydrogen. Solution-phase X-ray scattering and ultrafast transient optical spectroscopy analyses were used in tandem to correlate the self-assembled photocatalysts' structural integrity in solution with electron transfer and charge separation between the photosensitizer and catalyst fragments. Biphasic excited state decay kinetics were observed for several of the assemblies, suggesting that configurational dispersion plays a role in limiting photoinduced electron transfer. Notably, an assembly featuring a "push-pull" donor-photosensitizer-acceptor triad motif exhibits considerable ultrafast excited state quenching and, of the assemblies examined, presents the strongest opportunity for efficient solar energy conversion. These results will assist in the design and development of next-generation supramolecular photocatalyst architectures.

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

A Functional Model of Extradiol-Cleaving Catechol Dioxygenases: Mimicking the 2-His-1-Carboxylate Facial Triad

The synthesis and characterization of an iron-catecholate model complex of a tridentate 2-N-1-carboxylate ligand derived from L-proline are reported. The X-ray crystal structure of the complex [(L)(3)Fe(3)(DBC)(3)] (1) (where L is 1-(2-pyridylmethyl)pyrrolidine-2-carboxylate and DBC is the dianion of 3,5-di-tert-butyl catechol) reveals that the tridentate ligand binds to the iron center in a facial manner and mimics the 2-his-1-carboxylate facial triad motif observed in extradiol-cleaving catechol dioxygenases. The iron(III)-catecholate complex (1) reacts with dioxygen in acetonitrile in ambient conditions to cleave the C-C bond of catecholate. In the reaction, an equal amount of extra- and intradiol cleavage products are formed without any auto-oxidation product. The iron-catecholate complex is a potential functional model of extradiol-cleaving catechol dioxygenases.

Theoretical Investigation of Thermal Decomposition of Peroxidized Coelenterazines with and without External Perturbations

Thermal decomposition of peroxidized coelenterazines with and without external perturbations has been studied theoretically using the hybrid density functional theory (B3LYP) and the Coulomb-attenuating method (CAM). Possible roles of a hydrogen-bonding interface constituted by amino acid residues in the coelenterazine-biding site of aequorin are addressed by using simple model clusters with a polarizable continuum model to grasp some important aspects that may affect the electronic mechanism operating within the photoprotein. Calculations have revealed that the electronic property and stability of the peroxide are greatly affected by its protonation state and/or environmental effects, such as a polarizing medium and specific (localized) short-range electrostatic interactions, which may be critical for the bioluminescence activity. Theory highlights two mechanisms by which the neutral species can be activated, which otherwise decomposes by a homolytic O-O dissociation with a high barrier. In the first mechanism, the Tyr82-His16-Trp86 triad motif facilitates the deprotonation process of the phenolic OH group at the C(6) position of the coelenterazine and thereby makes it a sufficiently good electron donor to activate the O-O bond. In the second mechanism, intramolecular charge transfer is accomplished within the neutral peroxide by a proton delivery mediated via another triad motif, Tyr184-His169-Trp173, without the activation of the substrate itself. The combination of the first and second mechanisms leads to complete electron transfer for the formation of a radical pair as a local intermediate stabilized by the nearby triad motifs.

Purification and molecular characterization of a novel diadenosine 5′,5′′′-P 1,P 4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P 1,P 4-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K m values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.

A Second GDP-l-galactose Phosphorylase in Arabidopsis en Route to Vitamin C: COVALENT INTERMEDIATE AND SUBSTRATE REQUIREMENTS FOR THE CONSERVED REACTION

The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

AbstractExtensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val87‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.

Molecular and biological characterization of histidine triad protein in group A streptococci

Four Streptococcus pneumoniae genes, phtA, phtB, phtD, and phtE, as well as the slr gene of group A streptococci (GAS), encode proteins with a histidine triad motif (HxxHxH). Pht proteins function as protective antigens against S. pneumoniae infection. A search of the GAS genome database identified a novel protein, HtpA, possessing five histidine triad motifs. The htpA gene was shown to encode a 92.5-kDa protein located downstream of the fbaA and lbp genes, while Western blot analyses revealed that HtpA protein was expressed on the cell surfaces of all group A, B, C, and G streptococcal isolates tested. Immunization of mice with rHtpA induced antigen-specific antibody production and was effective after a single immunization, with antibody titers remaining constant for at least 84 days. In addition, HtpA-immunized mice survived after challenge with GAS strains isolated from patients with streptococcal toxic shock syndrome for significantly longer periods than sham-immunized mice. In that experiment, the HtpA-specific antibody was effectively induced by a single immunization and the specific antibody titer remained constant for at least 84 days. These results indicate that the novel histidine triad protein HtpA is a candidate vaccine for GAS infection.

Synthesis and characterization of amphiphilic o-phenylene ethynylene oligomers

We have previously reported the synthesis of short o-phenylene ethynylene oligomers with polar triethylene glycol side chains which adopt a helical conformation in solution with three residues per turn. Two new oligomers have been synthesized, a hexamer and a nonamer, incorporating a repeated triad motif of polar–nonpolar–polar sidechains in order to create a hydrophobic stripe in the folded conformation which we report here for the first time. Helical folding in solution was observed and, unlike the previously-reported oligomers, these new oligomers are ordered solids at room temperature. Although these oligomers were designed to assemble into helical bundle-like structures, no evidence for a quaternary-like structure was found. The difference in polarity between alkyl and triethylene glycol side chains is likely not strong enough to induce self-association of folded helices, especially since the molecules are not water soluble where the driving force for association of the nonpolar stripe would be larger. We expect that more polar side chains, granting water solubility, represent an important target for future research.