Involvement of Domain II in Toxicity of Anthrax Lethal Factor

Abstract

Anthrax lethal factor (LF) is a Zn2؉ -metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu 293 514 and Asn 516 resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, direct in vitro measurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.Anthrax toxin is derived from an exotoxin produced by the Gram-positive bacterium Bacillus anthracis. The toxin is composed of three proteins: protective antigen (PA), 1 edema factor (EF), and lethal factor (LF). PA, by itself, is not toxic; it serves to translocate EF and LF to the cytosol (1-4). Two cell surface receptors for PA (anthrax toxin receptor or ANTXR) have recently been identified (5,6). Following binding to ANTXR, PA is cleaved by cell surface-associated furin, removing a 20-kDa fragment and leaving a 63-kDa fragment (PA 63 ) attached to the ANTXR. This step is necessary to expose a binding site for EF or LF (7) as well as to remove steric hindrances to the subsequent oligomerization of PA into a heptamer (4,8,9). Following EF or LF binding to heptameric PA 63 , the toxin complex internalizes via the endosomal pathway (10 -12). The acidic environment of the endosome triggers a conformational change in the structure of PA, causing it to form a pore through which EF or LF apparently transits to the cytosol (4).EF is an adenylate cyclase (12). EF plus PA (edema toxin) is not lethal but causes edema when injected subcutaneously (13,14). LF is a Zn 2ϩ -metalloprotease that specifically cleaves the NH 2 termini of mitogen-activated protein kinase kinases (MEKs) 1 and 2 (15, 16), 3 (17) and 4, 6, and 7 (18), but not MEK 5 (18), resulting in their inactivation (15,19,20). Combinations of PA plus LF (lethal toxin) do not cause edema, but when injected intravenously rapidly induce hypotensive shock leading to death of the host (13,14).LF is a large 776-amino acid (90.2-kDa) protein (21). The crystal structure of LF has been solved to a resolution of 2.2 Å (22) (see Fig. 1). It is composed of four domains. Domain I comprises the NH 2 -terminal portion, which binds PA. Domain II (residues 263-297 and 385-550) shows structural similarity with the adenosine diphosphate-ribosylating toxin of Bacillus cereus but lacks the residues required for nicotinamide adenine dinucleotide binding and ca...

An Anthrax Lethal Factor-Neutralizing Monoclonal Antibody Protects Rats before and after Challenge with Anthrax Toxin

“…Acidic residues in domain III make on May 10, 2018 by guest http://iai.asm.org/ specific contact with the basic residues at the amino terminus of MAPKK (11). It has been presumed that LF-neutralizing antibody binds to domain I and thus directly inhibits the binding of LF to PA to neutralize LeTx (21,24).…”

“…Regarding the structure-function relationship of LF, domain I of LF binds to PA, and domains II, III, and IV together create a long catalytic groove into which the amino terminus of MAPKK fits (11,20). Acidic residues in domain III make on May 10, 2018 by guest http://iai.asm.org/ specific contact with the basic residues at the amino terminus of MAPKK (11).…”

Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

“…Lethal factor is a Zn 2+ -metalloprotease, which specifically cleaves the NH 2 -termini of MKK1, MKK2, MKK3, MKK4, MKK6, and MKK7, but not MKK5 (23)(24)(25). Cleavage results in loss of kinase function of MKKs (26). It has been shown that LeTx reverts the transformation of V12 H-ras-transformed NIH 3T3 cells in vitro and inhibits their growth and vascularization in vivo (27).…”

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

“…However, longer substrates can be cleaved more efficiently because of secondary interactions between LF and its substrate (10). We used phage display screening to determine the minimal length of substrate efficiently cleaved by LF and to identify substrate determinants responsible for optimal subsite occupancy in the LF-substrate interaction (19).…”

“…Briefly, 10 11 purified phages in phosphate-buffered saline (1.7 mM NaH 2 PO 4 , 5.2 mM Na 2 HPO 4 , and 150 mM NaCl, pH 7.4) were applied to each well and incubated for 1 h at 25°C. The wells were then washed three times in phosphate-buffered saline, 0.05% Tween 20, blocked with 1% bovine serum albumin, and incubated with LF in test wells and without LF in control wells.…”