The role of electrostatic charge in the membrane insertion of colicin A. Calculation and mutation.
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.
Lakey
J H
JH
Department of Biochemistry and Genetics, Medical School, University of Newcastle-upon-Tyne, England.
Parker
M W
MW
González-Mañas
J M
JM
Duché
D
D
Vriend
G
G
Baty
D
D
Pattus
F
F
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004563:Electrochemistry; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D003087:Colicins; D008081:Liposomes; D008563:Membrane Lipids