Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

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On mechanisms of colicin import: the outer membrane quandary

Biochemical Journal ◽

10.1042/bcj20180477 ◽

2018 ◽

Vol 475 (23) ◽

pp. 3903-3915 ◽

Cited By ~ 12

Author(s):

William A. Cramer ◽

Onkar Sharma ◽

S.D. Zakharov

Keyword(s):

Outer Membrane ◽

Catalytic Domain ◽

Efflux Pump ◽

Crystal Structure Analysis ◽

Multidrug Efflux Pump ◽

Terminal Domain ◽

Colicin E1 ◽

N Terminus ◽

T Domain

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10−9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

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Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The Journal of General Physiology ◽

10.1085/jgp.98.1.77 ◽

1991 ◽

Vol 98 (1) ◽

pp. 77-93 ◽

Cited By ~ 28

Author(s):

C K Abrams ◽

K S Jakes ◽

A Finkelstein ◽

S L Slatin

Keyword(s):

Voltage Dependence ◽

Ion Selectivity ◽

Lipid Vesicles ◽

Voltage Gating ◽

Site Directed Mutagenesis ◽

Phospholipid Bilayer ◽

Voltage Gated ◽

Colicin E1 ◽

Gating Charge ◽

Ion Conducting

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.

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An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: Effect on the structure and replication of the colicin E1 plasmid in vitro

MGG Molecular & General Genetics ◽

10.1007/bf00339004 ◽

1981 ◽

Vol 181 (1) ◽

pp. 52-56 ◽

Cited By ~ 18

Author(s):

Elisha Orr ◽

Walter L. Staudenbauer

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

B Subunit ◽

Coli Mutant ◽

Colicin E1

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Distinctions between Hydrophobic Helices in Globular Proteins and Transmembrane Segments as Factors in Protein Sorting

Journal of Biological Chemistry ◽

10.1074/jbc.m809017200 ◽

2008 ◽

Vol 284 (8) ◽

pp. 5395-5402 ◽

Cited By ~ 9

Author(s):

Fiona Cunningham ◽

Arianna Rath ◽

Rachel M. Johnson ◽

Charles M. Deber

Keyword(s):

Protein Sorting ◽

Globular Proteins ◽

Transmembrane Segments ◽

Hydrophobic Helices

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Seipin traps triacylglycerols to facilitate their nanoscale clustering in the ER membrane

10.1101/2020.10.26.355065 ◽

2020 ◽

Author(s):

Xavier Prasanna ◽

Veijo T. Salo ◽

Shiqian Li ◽

Katharina Ven ◽

Helena Vihinen ◽

...

Keyword(s):

Lipid Droplet ◽

In Silico ◽

Lipid Membrane ◽

Wild Type ◽

Binding Interface ◽

Biomolecular Simulations ◽

Hydrophobic Helices ◽

Tag Clustering ◽

Er Membrane ◽

In Cells

AbstractSeipin is a disk-like oligomeric ER protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While seipin-S166D mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

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