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. Phage-based 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.
Highlights
Anthrax is an infectious disease caused by the encapsulated, spore-forming bacterium Bacillus anthracis
Phage Display Library Screening and Substrate Selection— The conserved portion of the lethal factor (LF) substrate in MKKs consists of only eight amino acids (18), and even seven-amino acid sequences can serve as LF substrates (8)
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)
Summary
Materials, Bacterial Strains, and Vector DNA— Unless stated otherwise, chemicals were purchased from Sigma. Construction of Phage Display Libraries and Selection of Clones Cleaved by LF—Unless stated otherwise, the inserts for vector modification and library construction were prepared by annealing the indicated oligonucleotides (see supplemental Table I for oligonucleotide and peptide sequences), filling-in by Klenow fragment, and digesting with appropriate restriction enzymes. To prepare the second-iteration library, first an insert containing the double c-myc tag, the extended linker, and the LF target peptide was prepared from 2MycAscFor and 2MycAscRev oligonucleotides digested by ApaLI and XhoI and ligated into ApaLI- and XhoI-digested Fd-Base. This modification introduced an AscI site into the vector for subsequent library construction. LF15 is a substrate that was previously isolated from a synthetic peptide library (7) and is cleaved efficiently by LF
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