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

Abstract

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phagebased selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca 2؉ and Mn 2؉ . Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.Anthrax is an infectious disease caused by the encapsulated, spore-forming bacterium Bacillus anthracis. Systemic forms of the disease, such as inhalational anthrax, are characterized by nonspecific early symptoms, rapid progression, and lethality approaching 100% (1). The lethality of inhalational anthrax is high even with antibiotic treatment and is caused by accumulation of secreted anthrax toxin (2), which consists of three proteins as follows: protective antigen (PA), 2 lethal factor (LF), and edema factor. PA binds to membrane receptors, forms pore complexes, and translocates LF and edema factor into the host cell (3, 4). The PA⅐LF complex is known as the lethal toxin, a virulence factor with pleiotropic action that facilitates establishment of the B. anthracis infection. LF is a Zn 2ϩ -dependent metalloprotease related to the thermolysin family that cleaves mitogen-activated protein kinase kinases (5).Although the complete mechanism by which LF causes fatal intoxication is still unclear, inhibition of LF proteolytic activity may be an efficient means of preventing anthrax lethality. A better understanding of the LF catalytic mechanism will facilitate rational design and optimization of LF inhibitors with potential clinical applicability. Recent structural (6, 7), mechanistic (8), and in vivo studies (9, 10) of LF point to a sophisticated catalytic mechanism involving accurate recognition of multiple target substrates.Here we use substrate phage display and stopped-flow fluorimetry kinetics to examine both the substrate specificity and elementary steps of substrate processi...

The anthrax lethal factor and its MAPK kinase-specific metalloprotease activity

“…2B). Moreover, the fact that the replacement of Zn 2+ with Mn 2+ in the active site of tetanus neurotoxin and LF supports the proteolytic activity of both enzymes (Tonello et al 1997;Zakharova et al, 2009), whilst this is not the case for thermolysin (Holmquist and Vallee, 1974), is an additional evidence for a similar zinc coordination and mechanism of substrate hydrolysis.…”

“…An additional difference may be the affinity of the active site Zn 2+ which is not known, but appears to be higher in LF than in clostridial neurotoxins since it is impossible to completely remove the metal atom with chelating agents (Kochi et al 1994) as it was in tetanus and botulinum neurotoxins (Schiavo et al, 1992b,c). This could explain the little ''residual protease activity" of LF mutated at the Glu of the zinc-binding motif (Zakharova et al, 2009), though a residual contamination with bacterial proteases cannot be excluded. It should be noted that it would be hard to account for the successful two-hybrid search of the LF substrate if this Glu mutant would have been fully active (Vitale et al, 1998).…”

“…At this point, the enzyme-substrate complex may acquire a productive conformation that leads to the subsequent hydrolysis. This last passage is the rate-limiting step of the enzymatic reaction (Zakharova et al, 2009). In the active site, a water molecule forms a strong hydrogen bond with the Glu of the zinc coordinating motif (HExxH) and with the conserved Tyr-728.…”

“…By screening peptide libraries, Turk and colleagues identified a FRET peptide substrate of LF, which is very rapidly hydrolyzed . More recently, other peptide substrates efficiently cleaved by LF were selected from a phage display peptide library (Zakharova et al, 2009). Altogether, these LF peptide substrate studies lead to the conclusion that the most important determinants for a rapid hydrolysis by LF are an upstream basic cluster followed by an hydrophobic amino acid (++h in P5-P3 positions) and an hydrophobic residue at the P1 0 position.…”

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase

“…A promising approach to this end is phage display technology, which has been successfully developed during the past two decades for various enzymes [27], abzymes, antibodies [28e30], and peptides [31].…”