Involvement of Residues 147VYYEIGK153 in Binding of Lethal Factor to Protective Antigen of Bacillus anthracis Pankaj Gupta, 1 Aparna Singh, Vibha Chauhan, and Rakesh Bhatnagar 2 Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India Received November 20, 2000 Anthrax toxin is a complex of protective antigen (PA, 735 aa), lethal factor (LF, 776 aa), and edema factor (EF, 767 aa). PA binds to cell surface receptors and is cleaved by cell surface proteases into PA 63 , while LF and EF compete for binding to PA 63 . The PA 63 –LF/EF complex is internalized into the cytosol and causes different pathogenic responses in animals and cultured cells. 1–300 amino acid residues of LF have been viewed as the region responsible for the high affinity binding of LF to PA. Amino acid analysis of LF and EF revealed a common stretch of 7 amino acids (147VYYEIGK153). In the present study, each amino acid of this stretch was replaced by alanine at a time. Y148A, Y149A, I151A, and K153A mutants were found to be deficient in their ability to lyse J774A.1 cells and their binding ability to PA 63 was drastically reduced. We propose that these four amino acids play a crucial role in the process of binding of LF to PA 63 . © 2001 Academic Press Anthrax is a disease of herbivores caused by Bacillus anthracis, a Gram positive spore forming bacterium. Human infection is accidental and cases arise due to contact with the infected animals or animal products. Virulence of the bacterium is attributed to two factors, a poly D-glutamic acid capsule and a three component protein exotoxin. The genes coding for the toxin and the enzymes responsible for the capsule production are carried on B. anthracis plasmid pXO1 and pXO2 re- spectively (1, 2). The three proteins of the exotoxin are protective antigen (PA 83 kDa), lethal factor (LF 90 kDa), and edema factor (EF 89 kDa). None of the pro- teins are individually toxic but they combine pairwise to form lethal (PA + LF) and edema (PA + EF) toxins (3–5). PA acts as the common receptor binding moiety and it interacts with EF or LF to mediate their entry into the target cells (6, 7). PA binds to cell surface receptors where it is cleaved by furin like cellular pro- teases generating a cell bound, 63-kDa protein (PA 63 ) with a high affinity binding site for EF or LF (6). The complex is subsequently internalized by receptor- mediated endocytosis into endosomes (8, 9). Acidifica- tion of the endosomes brings about the oligomerization of PA 63 and is believed to play a crucial role in the process of translocation of LF/EF in the cytosol (10). Mouse peritoneal macrophages and macrophage like cell lines such as J774A.1 and RAW 264.7, etc. are sensitive to anthrax lethal toxin (11–13). Protein syn- thesis and calcium is required for expression of an- thrax lethal toxin activity in the macrophages (13, 14). Lethal toxin causes over production of certain cyto- kines such as IL-1 and TNF- in its target cells (15). There is activation of phospholipase C and protein kinase C in macrophages during cytolysis (16). LF acts as an endopeptidase and cleaves the amino terminus of mitogen activated protein kinase kinases 1and 2 (MAPKKs) and inhibits the MAPK signal transduction pathway. However the exact mechanism of cell death is not yet established (17). Amino acid analysis of LF and EF reveals an exten- sive homology in the residues 1–300 (18). Since both the proteins compete to bind to receptor bound, nicked PA and the intracellular actions and enzymatic activ- ities of these two proteins are different, the regions of homology have been viewed as the regions responsible for high affinity binding to PA (18 –20). Further insight into the amino acid sequence comparison of LF 1–250 and EF 1–250 using BESTFIT program of GCG revealed a stretch of 7 amino acids 147VYYEIGK153 which is present in lethal factor as well as edema factor. The aim of the study was to determine the importance of these seven residues in the process of binding of LF to PA. MATERIALS AND METHODS Reagents and supplies. The enzymes and chemicals used for DNA manipulation were purchased from Life Technologies (USA); 1 Present address: Dept. of Biological Sciences, Columbia Univer- sity, New York, NY. 2 To whom correspondence should be addressed. Fax: (91) 11-6165886/6198234/6169962. E-mail: rakesh@jnuniv.ernet.in or rakbhat@hotmail.com. Biochemical and Biophysical Research Communications 280, 158 –163 (2001) doi:10.1006/bbrc.2000.4099, available online at http://www.idealibrary.com on 158 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.