Book of Abstracts: Albany 2005
Prediction and Analysis of Complexes Formed Between the Anthrax Protective Antigen Heptameric ?Pre-Pore? and the Anthrax Lethal Factor and Between the Anthrax Lethal Factor and MAPKK-2 Peptide Substrate Models
Bacillus anthracis, a spore-forming infectious bacterium, produces a toxin consisting of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA) (1, 2). LF, the major anthrax toxin responsible for cell death, functions as a zinc-dependent protease to cleave mitogen-activated protein kinases. EF, when complexed with host cell calmodulin, becomes an adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion, while consuming cellular ATP. PA plays the critical role of facilitating entry of both EF and LF toxins into host cell cytoplasm. This endocytotic event is initiated following the interaction of PA monomers with anthrax toxin receptors present on the host cell membrane. Following the cleavage of a portion of the amino terminal domain 1 (∼ 20 kDa) of PA with the human protein furin, a heptameric ?cup-like pre-pore? arrangement of receptor-bound truncated PA monomers (Mr ∼ 63,000) is formed, which serves as a endocytotic transport vessel for EF and/or LF (3).
Here we report the results of two separate computational studies, one directed at predicting possible modes of interaction between the PA heptameric ?pre-pore? and LF, and another directed at understanding both steric and energetic requirements for effective interaction of LF with substrate analogs. Both computational efforts employed a variety of approaches, including ZDOCK (4) protein:protein docking simulations, RDOCK CHARMM refinement procedures (5), as well as MolProbity (6) and GRASP2 (7) analyses of final complexes. Our predicted refined ZDOCK structure for the PA heptamer:LF complex is in good agreement with experimental data (although no x-ray structure is currently available). ZDOCK also successfully predicted the wild-type structure for the complex between LF and the peptide substrate mimic VYPYPMEPT (1pwv.pdb). Docking studies carried out on mutant variants of both PA and LF in the two systems under study have indicated key residues in both anthrax toxins critical to the formation of effective complexes. For example, following the standard 3-stage RDOCK minimization protocol, the predicted wild-type PA:LF complex (shown below on the left and center) has a significantly lower energy than the mutant PA:LF complex (shown below on the far right), in which a set of negatively-charged PA residues facing the central core of the pre-pore have been mutated to alanine.
Gustaf H. Carlson School of Chemistry and Biochemistry
This study was supported by a research grant to D. J. N. from the National Institutes of Health (R15 AI054577-01).
References and Footnotes