Site-directed mutagenesis of the charged residues near the carboxy terminus of the colicin E1 ion channel

Shiver, John W.; Cohen, Fredric S.; Merrill, A. Rod

doi:10.1021/bi00422a019

Cited by 23 publications

(11 citation statements)

References 41 publications

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“…anchor for insertion of the colicin channel into the membrane has been discussed previously for colicin A (22) and for colicin El (7,13,27,(29)(30)(31). The tightness of the turn made by residues 489 to 493 does not seem to be a structural problem, as -5 residues can function as extramembrane turn regions connecting trans-membrane helices in bacteriorhodopsin (1,14).…”

Section: Discussionmentioning

confidence: 94%

“…The salt gradient (0 to 1 M NaCl) was programmed so that the column flow rate was 1 ml/min, 1 M NaCl was achieved in 30 min, and the mutant colicin was eluted at 0.3 M NaCl. Each mutant peak was collected in one or more fractions, and each fraction was run on a sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) Phast gel system (Pharmacia) to identify the most pure fraction.…”

Section: Methodsmentioning

confidence: 99%

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Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin

1991

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The paucity of crystallographic data on the structure of intrinsic membrane proteins necessitates the development of additional techniques to probe their structures. The colicin El ion channel domain contains one prominent hydrophobic region near its COOH terminus that has been proposed to be an anchor for the assembly of the channel. Saturation site-directed mutagenesis of the hydrophobic anchor region of the colicin El ion channel was used to probe whether it spanned the bilayer once or twice. A nonpolar amino acid was replaced by a charged residue in 29 mutations made at 26 positions in the channel domain. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic region and the periphery of the hydrophobic region caused a large decrease in the cytotoxicity of the purified mutant colicin El protein. This result implies that the hydrophobic domain spans the membrane bilayer twice in a helical hairpin loop, with the center of this domain residing in an aqueous or polar phase. The lengths of the trans-membrane helices appear to be approximately 18 and 16 residues. The absence of significant changes in ion selectivity in five of nine mutants indicated that these mutations did not cause a large change in the channel structure. The ion selectivity changes in four mutants and those previously documented for the flanking Lys residues imply that the hydrophobic hairpin is part of the channel lumen. Water may "abhor" the hydrophobic side of the channel, explaining the small effects of residue charge changes on ion selectivity.Colicin El is a bactericidal plasmid-encoded protein whose lethal action is a consequence of its ability to form a highly conductive, monovalent, voltage-gated ion channel in the Escherichia coli cytoplasmic membrane. The manner in which the membrane channel is assembled and gated is of interest for studies on the mechanism of action of toxins, protein import, and voltage-gated channels (8). Colicin El is one of six bactericidal channel-forming colicins (El, Ta, Tb, A, B, and N), the COOH-terminal channel domains of which have an appreciable level of primary sequence identity ( Fig. 1) (8, 24). The crystal structure determination of the watersoluble form of the 204-residue thermolytic channel peptide of colicin A, determined and refined to 0.25-nm resolution, revealed the presence of 10 a-helices (24). Helices 8 and 9, which are buried in the core of the soluble peptide, have been hypothesized to be inserted into the membrane bilayer as a hydrophobic a-helical hairpin, thereby providing an anchor for insertion of the other helices needed to form the final channel structure (7,24). This hypothesis has not yet been tested by topographical experiments, although the conclusion that Asp-473 of the channel resides near the cis side of the membrane (15) is consistent with a helical hairpin model.It is known that the COOH terminus of the channel is positioned on the cis side of the membrane (7, 38), that its secondary structure in the membrane is predominantly a...

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Section: Discussionmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin

1991

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“…Asolectin (Sigma) LUVs (ϳ20 mg/ml) were loaded with the Cl Ϫ -sensitive fluorophore, 6-methoxy-N-(3-sulfonopropyl) quinolinium (SPQ, 16 mM) (Molecular Probes, Eugene, OR) and were prepared according to the freeze-thaw technique as described previously (21,22), and the encapsulation buffer was 100 M KCl, 10 mM DMG, 1 mM CaCl 2 , 16 mM SPQ at pH 5.0. The phospholipid concentration was determined using the Bartlett assay for phosphorus as described by New (23).…”

Section: Methodsmentioning

confidence: 99%

“…Determination of Channel Peptide Cl Ϫ Efflux Activity-The in vitro channel activity of colicin E1 has been measured using a variety of techniques (21,33,34), and the channel activities of the WT and various mutant proteins are shown in Fig. 4B.…”

Section: Fig 1 Schematic Representation Of the Colicin E1 Ph Triggermentioning

confidence: 99%

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

Musse

Merrill

2003

Journal of Biological Chemistry

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The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, due to nutrient depletion or overcrowding, and these proteins often target-sensitive bacterial strains (1). The lethal action of colicins against their target cells is manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1-7). In this context, the bacterial machinery responsible for colicin biological activity features important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that possesses the naturally occurring colE1 plasmid; the holoprotein consists of three functional segments, the translocation, receptor-binding, and channel-forming domains. Initially, the receptor-binding domain (8) interacts with the vitamin B 12 receptor of target cells. Following recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm (9). In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state and then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The channel allows the passage of monovalent ions, resulting in the dissipation of the cationic gradients (H ϩ , K ϩ , and Na ϩ ) of the target cell, causing depolarization of the cytoplasmic membrane. In an effort to compensate for the membrane depolarization effected by the colicin E1 channel, Na ϩ /K ϩ -ATPase activity is increased in the host cell, resulting in the consumption of ATP reserves, without concomitant replenishment (4). The final outcome is host cell death.In addition to structural similarities between colicin E1 and several membrane-targeting proteins, an intriguing characteristic shared by colicin E1 and many membrane-active proteins is the observed requirement of an acidic environment for activation of the soluble structure in order to bind and insert into target cell membranes. The important effect of an acidic environment in conferring an optimum activity in vitro, and perhaps in vivo, for colicin E1 has been attributed to an onset of acid-induced protein unfolding events, resulting in an increased structural mobility/flexibility that may potentiate the complex sequence of structural changes of the channel peptide observed at the surface of a membrane (10 -12).An important contribution to our understanding of the mechanism of colicin E1 low pH activation was conducted by Merrill et al. (13) in which a precise pH-sensitive region with...

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“…8), there are several residues which can be mutated and which affect negatively charged residues. These include Glu468 (corresponds to Glu138 in colicin-A thermolytic fragment) [38] and Asp509 (Asp1 89) [39] which form the negatively charged dome in the calculated fields. Both mutated residues have little effect on in vivo activity but reduce the in vitro activity in vesicles and planar bilayers.…”

Section: The Role Of Asp78 In the Binding And Insertion Stepsmentioning

confidence: 99%

The role of electrostatic charge in the membrane insertion of colicin A

Lakey

Parker

González-Mañas

et al. 1994

European Journal of Biochemistry

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The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of PI 10.20 (colicin A has PI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.Proteins and peptides bind to lipid membranes because of electrostatic or hydrophobic forces. The hydrophobic interactions can depend either upon a hydrophobic region of the peptide or upon an attached fatty-acyl chain, whilst basic amino acid residues are often very important in electrostatic interactions because natural membranes are predominantly negatively charged. Proteins that bind purely electrostatically, such as cytochrome c [l] or protein kinase C [2] can be fully water soluble but should remain at the surface because they are not soluble in the membrane interior. Those proteins which use hydrophobic interactions can penetrate the lipid bilayer, but show reduced solubility in aqueous solution.Correspondence to J. H. Lakey, The Department of Biochemistry and Genetics, The Medical School, The University of Newcastleupon-Tyne, England NE2 4HHAbbreviations. D78N, mutant colicin-A protein in which aspartate at position 78 of the thermolytic fragment has been exchanged for an asparagine residue ; Br,,Ole,GroPGro, 1,2-bis-[(9,10)-di bromooleoyl]-sn-glycero-3-phospho-1 -sn-gly cerol ; Ole,GroPGro, 1,2-dioleoyI-sn-glycero-3-phospho-l -sn-glycerol ; Pam'GroPEtn, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; Dns-Pam'GroPEtn, N-(5-dimethyl-aminonaphthalene-I -sulfonyl)-l,2-dipalmitoylsn-glycero-3-phosphoethanolamine ; Myr,GroPGro, 1,2-dimyristoylsn-gl ycero-3-phospho-1 -sn-gl ycerol.The insertion of water-soluble proteins into membranes, which occurs in the cases of toxins [3, 41, C9 comple...

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Site-directed mutagenesis of the charged residues near the carboxy terminus of the colicin E1 ion channel

Cited by 23 publications

References 41 publications

Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin

Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

The role of electrostatic charge in the membrane insertion of colicin A

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