Identification of lysines within alpha 1-antichymotrypsin important for DNA binding. An unusual combination of DNA-binding elements.
The human serum serine protease inhibitor (serpin) alpha 1-antichymotrypsin (ACT) appears to be unique among serpins in its ability to bind to double-stranded DNA. Using site-directed mutagenesis and chemical modification, a tri-lysine sequence (residues 210-212) falling within a solvent exposed loop and the C-terminal peptide containing two lysines (residues 391 and 396) were shown to be important for DNA binding. Mutation of residues 210-212 from lysines to either glutamates or threonines abolished DNA binding. The Lys210-Thr211-212 and Thr210-Th4(211)-Lys212 variants displayed reduced affinity for DNA, especially at higher ionic strength. Limited acetylation of rACT with acetic anhydride led to loss of DNA binding and, conversely, DNA protected rACT from acetylation. A combination of CNBr digestion, peptide separation, and peptide sequencing identified Lys396, two residues from the C terminus, as the most reactive lysine in rACT. Acetylation of Lys396 is strongly decreased in the presence of DNA. The double mutant K391T/K396T-rACT had very little affinity for DNA. The epsilon-amines of lysines 210-212 are 8-15 A across a cleft from the epsilon-amines in Lys391 and Lys396, and together these two elements may form an unusual DNA binding domain. Attempts to isolate a DNA sequence to which ACT binds specifically have been unsuccessful to date, raising the possibility that nonspecific binding of ACT to DNA suffices to account for the ACT found in certain cell nuclei. ACT variants not binding to double-stranded DNA retain ACT protease inhibitory activity, a potentially important result for the use of ACT variants as therapeutic agents.
Naidoo
N
N
Department of Chemistry, University of Pennsylvania, Philadelphia 19104, USA.
Cooperman
B S
BS
Wang
Z M
ZM
Liu
X Z
XZ
Rubin
H
H
D000107:Acetylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000514:alpha 1-Antichymotrypsin / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D000514:alpha 1-Antichymotrypsin; D004247:DNA; D008239:Lysine