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Infection and Immunity, April 1999, p. 1860-1865, Vol. 67, No. 4
Oral Infection and Immunity Branch, National
Institute of Dental and Craniofacial Research, National Institutes
of Health, Bethesda, Maryland 20892
Received 10 November 1998/Returned for modification 9 December
1998/Accepted 14 January 1999
Anthrax toxin from Bacillus anthracis is a
three-component toxin consisting of lethal factor (LF), edema factor
(EF), and protective antigen (PA). LF and EF are the catalytic
components of the toxin, whereas PA is the receptor-binding component.
To identify residues of PA that are involved in interaction with the
cellular receptor, two solvent-exposed loops of domain 4 of PA (amino
acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the
altered proteins purified and tested for toxicity in the presence of
LF. In addition to the intended substitutions, novel mutations were
introduced by errors that occurred during PCR. Substitutions within the
large loop (aa 704 to 723) had no effect on PA activity. A mutated
protein, LST-35, with three substitutions in the small loop (aa 679 to
693), bound weakly to the receptor and was nontoxic. A mutated protein,
LST-8, with changes in three separate regions did not bind to receptor
and was nontoxic. Toxicity was greatly decreased by truncation of the
C-terminal 3 to 5 aa, but not by their substitution with nonnative
residues or the extension of the terminus with nonnative sequences.
Comparison of the 28 mutant proteins described here showed that the
large loop (aa 704 to 722) is not involved in receptor binding, whereas
residues in and near the small loop (aa 679 to 693) play an important
role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in
stabilizing a conformation needed for receptor-binding activity.
0019-9567/99/$04.00+0
Identification of a Receptor-Binding Region within
Domain 4 of the Protective Antigen Component of Anthrax Toxin

*
Corresponding author. Mailing address: Oral Infection
and Immunity Branch, National Institute of Dental and Craniofacial
Research, Bldg. 30, Room 316, 30 Convent Dr. MSC 4350, Bethesda, MD
20892-4350. Phone: (301) 594-2865. Fax: (301) 402-0396. E-mail: leppla{at}nih.gov.
Present address: Division of Nephrology, Mount Sinai School of
Medicine, New York, NY 10029-6574.
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