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-antichymotr ypsin (ACT) appears to be unique among serpins in its ability to bind to double-stranded DNA. Using site-directed mutagenesis and chemical m odification, a tri-lysine sequence (residues 210-212) falling within a solvent exposed loop and the C-terminal peptide containing two lysine s (residues 391 and 396) were shown to be important for DNA binding. M utation of residues 210-212 from lysines to either glutamates or threo nines abolished DNA binding. The Lys(210)-Thr(211)-Lys(212) and Thr(21 0)-Th4(211)-Lys(212) variants displayed reduced affinity for DNA, espe cially at higher ionic strength. Limited acetylation of rACT with acet ic anhydride led to loss of DNA binding and, conversely, DNA protected rACT from acetylation. A combination of CNBr digestion, peptide separ ation, and peptide sequencing identified Lys(396), two residues from t he C terminus, as the most reactive lysine in rACT. Acetylation of Lys (396) 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 Angstrom across a cleft from the epsilon-a mines in Lys(391) adn Lys(396), and together these two elements may fo rm an unusual DNA binding domain. Attempts to isolate a DNA sequence t o which ACT binds specifically have been unsuccessful to date, raising the possibility that nonspecific binding of ACT to DNA suffices to ac count for the ACT found in certain cell nuclei. ACT variants not bindi ng to double stranded DNA retain ACT protease inhibitory activity, a p otentially important result for the use of ACT variants as therapeutic agents.