The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity

Abstract

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4°C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K 1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4°C. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.