Interactions of colicin A domains with phospholipid monolayers and liposomes: relevance to the mechanism of action

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).     

A 136-amino-acid-residue COOH-terminal fragment of colicin A is endowed with ionophoric activity.

DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'STOP-Shine-Dalgarno-sequence-ATG' cassette encoding a termination codon, a Shine-Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH2-terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X-114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1-3 of the 204-amino-acid-residue colicin pore-forming domain (containing 10 alpha-helices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH-terminal domain.     

Isolation and molecular and functional properties of the amino-terminal domain of colicin A

A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A.     

Functional domains of colicin A

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed. From these results, we suggest that the NH₂-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor-binding domain. The COOH-terminal domain (residues 389 to 592) carries the pore-forming activity.     

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.     

pH-dependent membrane fusion is promoted by various colicins.

The ability of colicin A, a bacteriocin produced by some Enterobacteriaceae, to fuse phospholipid vesicles at acidic pH, was demonstrated by electron microscopy and resonance energy transfer. The fusion depends on protein concentration and on the nature of the phospholipids. Vesicles, prepared from Escherichia coli phospholipids, fused one or more rounds at pH 4.5 upon addition of stoichiometric amounts of colicin A. Fusion was not only induced by pore-forming colicins (E1, K) but also by colicins that contain nuclease activities (E2, E3). By recombinant DNA technology it is shown that the first glycine-rich 70 NH2-terminal amino acids and, most probably, the extreme COOH-terminal end of colicin A are involved in the fusion activity of the protein. The physiological relevance of this property of colicins is discussed.     

Interchain disulfide bonding in the regulatory subunit of cAMP-dependent protein kinase I

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.     

A yeast Golgi E-type ATPase with an unusual membrane topology.

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.     

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen.

Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH-terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2-terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. IX. Isolation and sequences of several large cyanogen bromide peptides.

The isolation and sequences of several large peptides from cyanogen bromide cleavage of the 1030-residue polypeptide chain of the NAD-specific glutamate dehydrogenase of Neurospora crassa are described. One of these is in the 669-residue sequence of the COOH-terminal end of the protein. The remaining peptides have been aligned in two partial sequences in the NH2-terminal portion of the polypeptide chain.     

A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion

The central region of the colicin A polypeptide chain has been fused to the N-terminal part of beta-lactamase through genetic recombination. This region comprising amino acid residues 70-335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of beta-lactamase.     

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.     

A molecular genetic approach to the functioning of the immunity protein to colicin A

Summary A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH₂-and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124–143 abolish the protecting function of Cai.     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.