Analysis of the membrane-binding domain of penicillin-binding protein 5 of Escherichia coli

Internal deletions close to the C-terminus of the Escherichia coli penicillin binding protein 5 (PBP5, DacA) have defined the C-terminal 18 residues of the protein as essential for membrane binding. This C-terminal sequence is capable of forming a strongly amphiphilic alpha-helix. In this paper we show that the PBP5 amphiphilic helix is able to anchor the periplasmic TEM-beta-lactamase to the inner membrane. In addition, we have demonstrated that mature PBP5 (lacking the N-terminal signal sequence) possesses the ability to bind to the membrane from a soluble form of the protein, showing that translocation across the membrane is unnecessary for anchoring to be established.     

An Aeromonas salmonicida gene which influences a-protein expression in Escherichia coli encodes a protein containing an ATP-binding cassette and maps beside the surface array protein gene.

A conserved Aeromonas salmonicida gene (abcA) affecting expression of the surface array protein gene (vapA) in Escherichia coli was identified. The 924-bp gene starts 205 bp after vapA and codes for a protein with a deduced molecular weight (M(r)) of 34,015 containing an N-terminal P-loop and significant homology to the ATP-binding cassette transport protein superfamily. AbcA was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by using T7 polymerase expression and DNA-directed translation and was copurified with the sarkosyl-soluble cytoplasmic membrane fraction. The protein displayed aberrant migration during SDS-PAGE. A lacZ fusion containing 128 bp of upstream sequence and 387 bases in the 5' end of abcA was constructed, and the beta-galactosidase activity of the abcA-lacZ fusion gene was shown to be similar in E. coli and A. salmonicida. The 130,000-M(r) AbcA-LacZ fusion protein was purified, and by using an ATP affinity column, the 129 AbcA N-terminal P-loop-containing residues were shown to bind ATP.     

Crystal structure of the tandem-type universal stress protein TTHA0350 from Thermus thermophilus HB8

The genome sequence of an extremely thermophilic bacterium, Thermus thermophilus HB8, revealed that TTHA0350 is a tandem-type universal stress protein (Usp) consisting of two Usp domains. Usp proteins, which are characterized by a conserved domain consisting of 130-160 amino acids, are inducibly expressed under a large number of stress conditions. The N-terminal domain of TTHA0350 contains a motif similar to the consensus ATP-binding one (G-2 x-G-9x-G-(S/T)), but the C-terminal one seems to lack the consensus motif. In order to determine its structural properties, we determined the crystal structures of TTHA0350 in the unliganded form and TTHA0350?2ATP at 2.50 and 1.70 ? resolution, respectively. This is the first structure determination of a Usp family protein in both unliganded and ATP-liganded forms. TTHA0350 is folded into a fan-shaped structure which is similar to that of tandem-type Usp protein Rv2623 from Mycobacterium tuberculosis. However, the dimer assembly with C2-symmetry in TTHA0350 is quite different from that with D2-symmetry in Rv2623. The X-ray structure showed that not only the N-terminal but also the C-terminal domain binds one ATP, although the ATP-binding motif could not be detected in the C-terminal domain. The loop interacting with ATP in the C-terminal domain is in a conformation quite different from that in the N-terminal domain.     

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.     

Oligomerization of the chromatin-structuring protein H-NS

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.     

Influence of lipids on membrane assembly and stability of the potassium channel KcsA

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two-dimensional 1H-nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long alpha-helix containing eight turns along nearly the full length of the peptide except for four N-terminal residues and six C-terminal residues. The electrostatic surface map shows that the N-terminal segment of the alpha-helix, residues 5-22, is an amphipathic alpha-helix with a clear separation of hydrophobic and hydrophilic faces, and that the C-terminal segment of the alpha-helix, residues 23-36, is a hydrophobic alpha-helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N-terminal segment of the alpha-helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.     

Characterization of a low molecular weight protein of the ATP synthetase complex from beef heart and rat liver mitochondria with a high affinity monoclonal antibody

A monoclonal antibody raised against beef heart mitochondria elicited a strong reaction on Western Blot with a 16 kD protein in preparations of beef heart mitochondria, ammonia particles, oligomycin sensitive ATPase and Complex V, in addition to showing a lesser affinity for the partially purified 30 kD ADP/ATP carrier. The antibody also reacted with a 17 kD protein in rat liver mitochondria and an enriched membrane vesicle fraction. The N-terminal sequence of the first twenty amino acids of both the beef heart and rat liver proteins contained significant homology. Comparison with results in the literature indicate that the proteins represent the delta subunit of the ATP synthetase complex. Further evidence suggests that the epitope for the antibody may reside at the C-terminal 30-40 amino acid residues of both proteins.     

The DNA helicase, Hmi1p, is transported into mitochondria by a C-terminal cleavable targeting signal.

We have identified a novel mitochondrial targeting signal in the precursor of the DNA helicase Hmi1p of Saccharomyces cerevisiae that is located at the C terminus of the protein. Similar to classical N-terminal presequences, this C-terminal targeting signal consists of a stretch of positively charged amino acids that has the potential to form an amphipathic alpha-helix. Deletion of the C-terminal 36 amino acids of helicase resulted in loss of import into mitochondria, while deletion of the N-terminal 40 amino acids had no effect. When C-terminal regions of the helicase were placed at the C terminus of a passenger protein, dihydrofolate reductase, the resulting fusion proteins were directed into the mitochondrial matrix, and the C-terminal region of helicase became proteolytically processed. Import of helicase occurs in a C- to N-terminal direction; it requires a membrane potential and the TIM17-23 translocase together with mitochondrial Hsp70. Helicase is the only mitochondrial matrix protein identified thus far with a cleavable targeting signal at its C terminus.     

Effects of single amino acid substitution on the collision-induced dissociation of intact protein ions: Turkey ovomucoid third domain

Expanded understanding of the factors that direct polypeptide ion fragmentation can lead to improved specificity in the use of tandem mass spectrometry for the identification and characterization of proteins. Like the fragmentation of peptide cations, the dissociation of whole protein cations shows several preferred cleavages, the likelihood for which is parent ion charge dependent. While such cleavages are often observed, they are far from universally observed, despite the presence of the residues known to promote them. Furthermore, cleavages at residues not noted to be common in a variety of proteins can be dominant for a particular protein or protein ion charge state. Motivated by the ability to study a small protein, turkey ovomucoid third domain, for which a variety of single amino acid variants are available, the effects of changing the identity of one amino acid in the protein sequence on its dissociation behavior were examined. In particular, changes in amino acids associated with C-terminal aspartic acid cleavage and N-terminal proline cleavage were emphasized. Consistent with previous studies, the product ion spectra were found to be dependent upon the parent ion charge state. Furthermore, the fraction of possible C-terminal aspartic acid cleavages observed to occur for this protein was significantly larger than the fraction of possible N-terminal proline cleavages. In fact, very little N-terminal proline cleavage was noted for the wild-type protein despite the presence of three proline residues in the protein. The addition/removal of proline and aspartic acids was studied along with changes in selected residues adjacent to proline residues. Evidence for inhibition of proline cleavage by the presence of nearby basic residues was noted, particularly if the basic residue was likely to be protonated.     

Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.

Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.     

Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action.

Pediocin-like antimicrobial peptides (AMPs) form a group of lactic acid bacteria produced, cationic membrane-permeabilizing peptides with 37 to 48 residues. Upon exposure to membrane-mimicking entities, their hydrophilic, cationic, and highly conserved N-terminal region forms a three-stranded antiparallel beta-sheet supported by a conserved disulfide bridge. This N-terminal beta-sheet region is followed by a central amphiphilic alpha-helix and this in most (if not all) of these peptides is followed by a rather extended C-terminal tail that folds back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal half. There is a flexible hinge between the beta-sheet N-terminal region and the hairpin C-terminal region and one thus obtains two domains that may move relative to each other. The cationic N-terminal beta-sheet domain mediates binding of the pediocin-like AMPs to the target-cell surface through electrostatic interactions, while the more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the hydrophobic part of the target-cell membrane, thereby mediating leakage through the membrane. The hinge provides the structural flexibility that enables the C-terminal hairpin domain to dip into the hydrophobic part of the membrane. Despite extensive sequence similarities, these AMPs differ markedly in their target-cell specificity, and results obtained with hybrid AMPs indicate that the membrane-penetrating hairpin-like C-terminal domain is the major specificity determinant.Bacteria that produce pediocin-like AMPs also produce a 11-kDa cognate immunity protein that protects the producer. The immunity proteins are well-structured, 4-helix bundle cytosolic proteins. They show a high degree of specificity in that they largely recognize and confer immunity only to their cognate AMP and in some cases to a few AMPs that are closely related to their cognate AMP. The C-terminal half of the immunity proteins contains a domain that is involved in specific recognition of the C-terminal membrane-penetrating specificity-determining hairpin domain of the cognate AMP.     

The nucleotide-binding site of HisP, a membrane protein of the histidine permease. Identification of amino acid residues photoaffinity labeled by 8-azido-ATP

The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed.     

The solution structure of frenatin 3, a neuronal nitric oxide synthase inhibitor from the giant tree frog, Litoria infrafrenata

The peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind amphipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an amphipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region.     

Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing.

A single gene, VMA1, encodes the 69-kDa subunit of the vacuolar membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae. We have proposed that the subunit is synthesized as a precursor of 120 kDa (1,071 amino acids) and then converted to the 69-kDa form by an unusual processing reaction, which removes the internal domain of 454 amino acids (residues 284-737) and joins the N- and C-terminal domains. Cysteine to serine mutations at residues 284 and 738, the residues that bracket the internal domain, were introduced into the VMA1 gene by site-directed mutagenesis, and the mutant genes were expressed in a null vma1 mutant. Cells harboring either of the mutant vma1 genes accumulate nonfunctional fragments of the subunit. The mutation of Cys-284 inhibited the cleavage of the N-terminal junction site. Cys-738-->Ser mutation appeared to block the processing at both junction sites although the mutant gene yielded a small fraction of the functional 69-kDa subunit.     

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.     

Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells

A split NanoLuc assay system consisting of two fragments, large N-terminal and small C-terminal regions (NanoBiT), was developed to investigate protein-protein interactions within living cells. Interestingly, the replacement of five amino acids among 11 C-terminal amino acids dramatically increased affinity against the large N-terminal fragment, LgBiT, and the complex had NanoLuc luciferase activity. In this study, we first applied this small fragment, HiBiT, to elucidate the expression of ATF4 protein by transient overexpression of HiBiT-tagged ATF4. According to the regulation of intrinsic ATF4 protein, stabilization of HiBiT-tagged ATF4 with a proteasome inhibitor, MG132, was observed by detecting luciferase activity in cell lysate and after SDS-PAGE and transfer onto a PVDF membrane. Next, we knocked-in the HiBiT-epitope tag into the ATF4 gene using the CRISPR/Cas9 system and rapidly selected positive clones by measuring luciferase activity in an aliquot of each cell suspension. Using a selected clone, we observed that the expression of HiBiT-tagged ATF4 in the selected cells varied in response to treatment with protein synthesis inhibitors or proteasome inhibitors and tunicamycin. Altogether, this novel HiBiT tag is a useful tool to evaluate the endogenous expression levels of proteins of interest.     

A membrane protein, EzrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ

FtsZ polymerizes to form a dynamic ring structure called the Z-ring at the midcell of bacteria. EzrA, a membrane protein, has been shown to prevent the formation of aberrant Z-rings in the low GC Gram-positive bacteria by inhibiting FtsZ assembly. In this study, we show that Bacillus subtilis (B. subtilis) EzrA inhibited the assembly and bundling of B. subtilis FtsZ. It increased the critical concentration of FtsZ assembly and depolymerized the preformed FtsZ polymers in vitro. We obtained evidence suggesting that B. subtilis EzrA forms complex with B. subtilis FtsZ in vitro. EzrA was found to bind to FtsZ at a single site with a dissociation constant of 4.3 +/- 0.6 microM. EzrA-FtsZ interaction has a significant electrostatic contribution as apparent from the effect of salt on their binding interactions. To elucidate the site of interaction between EzrA and FtsZ, we deleted 16 amino acid residues from the extreme C-terminal tail of B. subtilis FtsZ, which are conserved in FtsZ orthologues. EzrA did not inhibit the assembly of C-terminal truncated B. subtilis FtsZ. It also did not bind to the C-terminal truncated FtsZ detectably, suggesting that EzrA interacts with FtsZ through its conserved C-terminal tail residues. Further, a 17-residue synthetic peptide (365-382) of the C-terminal tail of FtsZ (CTP17) was used to probe the interaction of EzrA with the C-terminal tail of FtsZ. CTP17 bound to EzrA, inhibited the binding of EzrA to FtsZ, and surmounted the inhibitory effects of EzrA on the assembly of FtsZ in vitro. The data together showed that EzrA binds to the C-terminal tail of FtsZ. FtsA, a positive regulator of FtsZ assembly, is also known to interact with the C-terminal tail of FtsZ. The results indicated an interesting possibility that the assembly dynamics of FtsZ in the Z-ring is regulated by the competition between positive and negative regulators sharing the same binding site on FtsZ.     

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.     

Lysine-dependent multipoint binding of the Borrelia burgdorferi virulence factor outer surface protein E to the C terminus of factor H

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18-20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala(151)-Lys(166)) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.