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Identification of the minimum segment in which the threonine 246 residue is a potential phosphorylated site by protein kinase A for the LukS‐specific function of staphylococcal leukocidin
Author(s) -
Nariya Hirofumi,
Nishiyama Akihito,
Kamio Yoshiyuki
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01100-9
Subject(s) - leukocidin , mutant , threonine , biology , panton–valentine leukocidin , peptide sequence , microbiology and biotechnology , hemolysin , residue (chemistry) , biochemistry , staphylococcus aureus , virulence , serine , phosphorylation , methicillin resistant staphylococcus aureus , gene , genetics , bacteria
Staphylococcal leukocidin and γ‐hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for γ‐hemolysin. In this report, we identify the minimum segment responsible for the LukS‐specific function of leukocidin. After chemical analysis and homology study of the amino acid sequence of the C‐terminal region between LukS and Hlg2, we found a unique 5‐residue sequence I 242 K 243 R 244 S 245 T 246 in LukS in which the 4‐residue KRST is identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. To elucidate whether the 5‐residue segment is essential for the LukS function, we created plasmids containing a series of mutant genes corresponding to the 5‐residue sequence and expressed them in Escherichia coli . The mutant proteins were purified and assayed for their leukocytolytic activity with LukF. The mutant MLS‐TS, in which the T 246 in the 5‐residue sequence was replaced by S, showed leukocidin activity 10 times higher than that of the intact LukS. However, neither mutant MLS‐TY nor MLS‐TA, in which T 246 was replaced by Y or A, respectively, showed leukocidin activity. The 5‐residue segment was found to be deleted in Hlg2. The mutant of Hlg2, in which the 5‐residue segment was inserted at the position that the segment is deleted, showed leukocidin activity. The boiled LukS, MLS‐TS, and MHS‐Z were strongly phosphorylated with [γ‐ 32 P]ATP in the presence of protein kinase A in a cell‐free system. Thus, we conclude that the 5‐residue segment I 242 K 243 R 244 S 245 T 246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.