Involvement of conserved histidine, lysine and tyrosine residues in the mechanism of DNA cleavage by the caspase-3 activated DNase CAD

Abstract

The caspase-activated DNase (CAD) is involved in DNA degradation during apoptosis. Chemical modification of murine CAD with the lysine-specific reagent 2,4,6-trinitrobenzenesulphonic acid and the tyrosine-specific reagent N-acetylimidazole leads to inactivation of the nuclease, indicating that lysine and tyrosine residues are important for DNA cleavage by this enzyme. The presence of DNA or the inhibitor ICAD-L protects the enzyme from modification. Amino acid substitution in murine CAD of lysines and tyrosines conserved in CADs from five different species leads to variants with little if any catalytic activity, but unaltered DNA binding (K155Q, K301Q, K310Q, Y247F), with the exception of Y170F, which retains wild-type activity. Similarly, as observed for the previously characterised H242N, H263N, H308N and H313N variants, the newly introduced His-->Asp/Glu or Arg exchanges lead to variants with <1% of wild-type activity, with two exceptions: H313R shows wild-type activity, and H308D at pH 5.0 exhibits approximately 5% of wild-type activity at this pH. Y170F and H313R produce a specific pattern of fragments, different from wild-type CAD, which degrades DNA non-specifically. The recombinant nuclease variants produced in Escherichia coli were tested for their ability to form nucleolytically active oligomers. They did not show any significant deviation from the wild-type enzyme. Based on these and published data possible roles of the amino acid residues under investigation are discussed.

Figure 1

Chemical modification of murine CAD by TNBS and NAI. Free GST-mCAD was modified by TNBS (top) and NAI (bottom) for the indicated time (1.5, 3, 6, 7.5 and 9 min). Subsequently the residual nucleolytic activities of the aliquots withdrawn from the modification reaction mixtures at the indicated time points were measured by a plasmid DNA cleavage assay (10 min). TNBS and NAI inactivate free GST-mCAD in a time- and dose-dependent manner. (ST, length standard; S, substrate DNA; C, control reaction; oc, open circular DNA; lin, linear DNA; sc, supercoiled DNA.)

Figure 2

Mutational analysis of variants with exchanged conserved lysine and tyrosine residues of GST-mCAD. (A) Amino acid residues Lys155, Lys301 and Lys310 as well as Tyr170 and Tyr247, which were exchanged to glutamine or phenylalanine, respectively, are fully conserved among the apoptotic nucleases known from the five indicated species [Mus musculus (GenBank accession nos AB009377, NM_007859), Rattus norvegicus (GenBank accession no. AF136598), Homo sapiens (GenBank accession nos AF064019, AF039210, AB013918, NM_004402), Danio rerio (GenBank accession no. AF286179) and Drosophila melanogaster (GenBank accession nos AF149797, AB036773)]. Shading is according to the Blosum 62 scoring matrix, with black shading for 100%, dark grey for 80% and light grey for 60% conserved amino acid residues. (B) SDS–PAGE analysis of variants of GST-mCAD that were produced as a complex with hICAD-L in E.coli and activated by treating the complex with recombinant caspase-3. (C) DNA cleavage activity of the GST-mCAD variants were measured by the disappearance of supercoiled plasmid DNA, analysed by agarose gel electrophoresis. GST-mCAD-Y170F is the only variant that retains wild-type activity with respect to the cleavage of supercoiled DNA, whereas all other variants exhibit strongly reduced cleavage activities. GST-mCAD-Y170F produces a pattern of fragments of defined length instead of randomly cut fragments appearing as a ‘smear’ (see wild-type GST-mCAD). Note the different concentrations of the variants as indicated.

Figure 3

Residual DNA cleavage activities of variants of GST-mCAD with His→Asp/Glu substitutions. Variant H308D is the only variant with a measurable activity at pH 5.0. All other variants, except wild-type GST-mCAD, are virtually inactive at this low pH.

Figure 4

DNA binding by the GST-mCAD variants. DNA binding of the GST-mCAD variants was investigated using a DNA–cellulose binding assay (16). Caspase-3-treated GST-CAD/hICAD-L complex (2.5 µg) and GST (2.5 µg) were incubated with 50 µl of a DNA–cellulose suspension. After washing, bound proteins were eluted by incubating the suspension for 5 min at 95°C in 10 µl SDS-gel loading buffer and subsequently analysed by SDS–PAGE. As can be seen, all GST-mCAD variants bind to DNA–cellulose, whereas GST alone does not.

Figure 5

Variants of CAD with reduced catalytic activity. (A) Inohara et al. (11) and Otomo et al. (14) have identified three conserved lysine residues (Lys12, Lys21 and Lys35) in the N-terminal domain of CAD (amino acids 1–83) that contribute to ICAD-L binding. Inohara et al. (11) have also identified three conserved residues (Gly55, Phe63 and Trp81) in the N-terminal domain of CAD that seem to enhance the nuclease activity of CAD without being essential for catalysis. According to their results, the catalytic nuclease domain is located in the C-terminal region of the protein (amino acids 84–344), since the Δ1–83 variant of CAD exhibits a moderate nucleolytic activity, whereas mutant forms with deletions in the C-terminal domain (Δ290–344, Δ162–344 and Δ84–344) have no nuclease activity. It must be emphasised that Otomo et al. (14) have reported contradictory results, according to which the C-terminal domain of CAD (Δ1–83) does not show any nucleolytic activity. (B) The C-terminal domain of CAD contains several histidine, lysine and tyrosine residues, which have been identified as being catalytically relevant by chemical modification and alignment-guided site-directed mutagenesis (16,17; this work). Variants of CAD with substitutions of the amino acid residues marked with an asterisk (Tyr170, His242, His308 and His313) are active but produce a defined cleavage pattern when hydrolysing DNA. (C) It can be concluded from the structural studies as well as the chemical modification and mutational analyses that the region of CAD interacting with ICAD-L, besides the known interaction of the N-terminal domain (12–15), includes the amino acid residues of CAD up to residue 241, whereas the DNA binding site is between amino acid residues Lys155 and His313.