Ligands of Thermophilic ABC Transporters Encoded in a Newly Sequenced Genomic Region of Thermotoga maritima MSB8 Screened by Differential Scanning Fluorimetry

The chromosome of Thermotoga maritima strain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 μM, 0.300 μM, and 56.78 μM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound with K_d (dissociation constant) values of 0.042 μM, 0.059 μM, and 1.436 μM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars: T. maritima MSB8 genomovar TIGR and T. maritima MSB8 genomovar DSM 3109, respectively.     

Ligand Binding Turns Moth Pheromone-binding Protein into a pH Sensor: EFFECT ON THE ANTHERAEA POLYPHEMUS PBP1 CONFORMATION*

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the “open” (bound) conformation at high pH but switches to the “closed” (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of ∼1:1. Mutation of His⁷⁰ and His⁹⁵ to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.     

Identification of Ribosomal Protein L1-Binding Sites in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis> mRNAs

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.     

Ligand-induced conformational changes in a thermophilic ribose-binding protein

Background

Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.

Results

Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation ( ^app T _m value is 108°C) than the mesophilic Escherichia coli homolog (ecRBP) ( ^app T _m value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.

Conclusion

Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.     

High-resolution structure of shikimate dehydrogenase from Thermotoga maritima reveals a tightly closed conformation

Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 Å by molecular replacement. SDH from T. maritima showed a monomeric architecture. The overall structure of SDH from T. maritima comprises the N-terminal α/β sandwich domain for substrate binding and the C-terminal domain for NADP binding. When the T. maritima SDH structure was compared with those of the SDHs from other species, the SDH from T. maritima was in a tightly closed conformation, which should be open for catalysis. Notably, α7 moves toward the active site (∼5 Å), which forces the SDH of T. maritima in a more closed form. Four ammonium sulfate (AMS) ions were identified in the structure. They were located in the active site and appeared to mimic the role of the substrate in terms of the enzyme activity and stability. The new high resolution structural information reported in this study, including the AMS binding sites as a potent inhibitor binding site of SDHs, is expected to supplement the existing structural data and will be useful for structure-based antibacterial discovery against SDHs.     

The 5.5 Protein of Phage T7 Inhibits H-NS through Interactions with the Central Oligomerization Domain

The 5.5 protein (T7p32) of coliphage T7 (5.5_T7) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5_T7 protein is insoluble when expressed in Escherichia coli, but we find that 5.5_T7 can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5_T7 binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5_T7 can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5_T7 protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5_T7 protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression.     

Crystal Structures of an Oligopeptide-Binding Protein from the Biosynthetic Pathway of the β-Lactamase Inhibitor Clavulanic Acid

Clavulanic acid (CA) is a clinically important β-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 Å resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.     

Structural Adaptations that Modulate Monosaccharide, Disaccharide, and Trisaccharide Specificities in Periplasmic Maltose-Binding Proteins

Periplasmic binding proteins comprise a superfamily that is present in archaea, prokaryotes, and eukaryotes. Periplasmic binding protein ligand-binding sites have diversified to bind a wide variety of ligands. Characterization of the structural mechanisms by which functional adaptation occurs is key to understanding the evolution of this important protein superfamily. Here we present the structure and ligand-binding properties of a maltotriose-binding protein identified from the Thermus thermophilus genome sequence. We found that this receptor has a high affinity for the trisaccharide maltotriose (K_d < 1 μM) but little affinity for disaccharides that are transported by a paralogous maltose transport operon present in T. thermophilus. Comparison of this structure to other proteins that adopt the maltose-binding protein fold but bind monosaccharides, disaccharides, or trisaccharides reveals the presence of four subsites that bind individual glucose ring units. Two loops and three helical segments encode adaptations that control the presence of each subsite by steric blocking or hydrogen bonding. We provide a model in which the energetics of long-range conformational equilibria controls subsite occupancy and ligand binding.     

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.     

Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide-binding protein.

Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide-binding protein. Similar studies performed on the maltose-binding and galactose-binding proteins revealed centrally positioned binding frames of approximately 160 aminoacyl residues. The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein. We propose modes of binding to account for the data.     

Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 Å, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.     

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe⁷⁷ and Tyr²⁷⁵, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe⁷⁷ restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr²⁷⁵ specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.     

Binding properties of pheromone-binding protein 1 from the common cutworm Spodoptera litura

Pheromone-binding proteins (PBPs) were formerly thought to act as passive pheromone carriers. However, recent studies, particularly in Drosophila melanogaster, suggest that PBPs are involved in the recognition of semiochemicals, thus making ligand-binding studies more meaningful. Previously, we cloned three PBPs from Spodoptera litura (Slit), and showed that SlitPBP1 is much more abundant than the other two, particularly in male antennae. To investigate the ligand specificity of SlitPBP1, we expressed the protein in a bacterial system and performed binding experiments with the three components of the specific sex pheromones (Z9-14:Ac, Z9,E11-14:Ac and Z9,E12-14:Ac), as well as with 26 volatile ligands. The results indicated that SlitPBP1 bound all three sex pheromone components with dissociation constants between 0.6 and 1.1 μM. The same protein also bound with comparable affinities several pheromone analogs, but not plant volatiles. The presence of a double bond was the most important element for a strong binding, while its position and configuration also affected the affinity. Finally, the binding of pheromone components is strongly affected by pH, showing a critical pH value corresponding to isoelectric point of the protein. This suggests that a pH-dependent conformational mechanism might exist in SlitPBP1 for pheromone binding and release.     

Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.     

Phosphorylation-dependent protein interaction with Trypanosoma brucei 14-3-3 proteins that display atypical target recognition

Background

The 14-3-3 proteins are structurally conserved throughout eukaryotes and participate in protein kinase signaling. All 14-3-3 proteins are known to bind to evolutionally conserved phosphoserine-containing motifs (modes 1 and/or 2) with high affinity. In Trypanosoma brucei, 14-3-3I and II play pivotal roles in motility, cytokinesis and the cell cycle. However, none of the T. brucei 14-3-3 binding proteins have previously been documented.

Methodology/Principal Findings

Initially we showed that T. brucei 14-3-3 proteins exhibit far lower affinity to those peptides containing RSxpSxP (mode 1) and RxY/FxpSxP (mode 2) (where x is any amino acid residue and pS is phosphoserine) than human 14-3-3 proteins, demonstrating the atypical target recognition by T. brucei 14-3-3 proteins. We found that the putative T. brucei protein phosphatase 2C (PP2c) binds to T. brucei 14-3-3 proteins utilizing its mode 3 motif (–pS/pTx_1-2-COOH, where x is not Pro). We constructed eight chimeric PP2c proteins replacing its authentic mode 3 motif with potential mode 3 sequences found in Trypanosoma brucei genome database, and tested their binding. As a result, T. brucei 14-3-3 proteins interacted with three out of eight chimeric proteins including two with high affinity. Importantly, T. brucei 14-3-3 proteins co-immunoprecipitated with an uncharacterized full-length protein containing identified high-affinity mode 3 motif, suggesting that both proteins form a complex in vivo. In addition, a synthetic peptide derived from this mode 3 motif binds to T. brucei 14-3-3 proteins with high affinity.

Conclusion/Significance

Because of the atypical target recognition of T. brucei 14-3-3 proteins, no 14-3-3-binding proteins have been successfully identified in T. brucei until now whereas over 200 human 14-3-3-binding proteins have been identified. This report describes the first discovery of the T. brucei 14-3-3-binding proteins and their binding motifs. The high-affinity phosphopeptide will be a powerful tool to identify novel T. brucei 14-3-3-binding proteins.     

The Bacterial Chromatin Protein HupA Can Remodel DNA and Associates with the Nucleoid in Clostridium difficile

The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA and modulate its conformation, and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of Clostridium difficile have been characterized to date.Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10-kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G + C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.     

The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.