Volume 271, Number 47,
Issue of November 22, 1996
pp. 29752-29758
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Electrostatic Interactions in Defining the Potency of
Neurotoxin B-IV from Cerebratulus lacteus
(Received for publication, July 17, 1996, and in revised form, September 5, 1996)
Paul H.
Wen
and
Kenneth M.
Blumenthal
From the Department of Molecular Genetics, Biochemistry and
Microbiology, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0524
Chemical modification implicates arginine residues of
the Cerebratulus lacteus neurotoxin B-IV in biological
activity. In the present study, we used site-directed mutagenesis to
assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in
this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as
natural toxin. Circular dichroism spectroscopy indicates that there are
no major conformational changes in any of these muteins. These results
therefore demonstrate the requirement for a guanidinium group at
position 17, and a positive charge at position 25. NMR analyses
(Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992)
Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV
to contain two antiparallel 
-helices, which together include 57% of
the sequence. Both Arg-17 and Arg-25 lie on the same face of the
N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13
and Asp-21. However, charge neutralizing mutations of the latter two
sites have no effects on biological activity. Arg-34, situated near the
N terminus of helix 2 (residues 33-49) is also important for activity,
as its replacement by Gln or Ala diminishes activity by 20- and
80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal
denaturation experiments suggest that R34Q may be structurally
destabilized relative to wild-type B-IV.
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