Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence

Abstract

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gtn 9, Arg 41, p r 76, Arg 1 1 1 , Asn 170, Tyr 175, and Tyr 21 1 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn*+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.Keywords: DNase I; protein-DNA interactions; site-directed mutagenesis; structure-function analysis Human deoxyribonuclease (DNase I), a pancreatic endonuclease that catalyzes the hydrolysis of double-stranded DNA, is currently used for the treatment of pulmonary complications in cystic fibrosis patients (Ramsey, 1996). The recombinant human enzyme is inhaled into the airways where it degrades DNA to lower molecular weight forms, thus reducing the viscoelasticity of CF sputum and improving lung function (Fuchs et al., 1994, Ramsey et al., 1993. In addition, the use of DNase I in a murine model of systemic lupus erythematosus (SLE) has been investigated with encouraging results (Macanovic et al., 1996). The pharmacological and clinical significance of DNase I has led us to recently engineer actin-resistant Reprint requests to: Robert A. Lazarus, Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080: e-mail: lazarus.bob@gene.com.Abbreviations: DNase I, deoxyribonuclease I; CF, cystic fibrosis; SLE, systemic lupus erythematosus: EDTA, ethylenediamine tetraacetic acid: TBE, tris-borate EDTA: Hepes, N-2-hydroxylethylpiperazine-N'-2-ethanesulfonic acid; SMase, sphingomyelinase. One or three letter codes are used to represent naturally occurring L-amino acids. When referring to mutants the wild-type amino acid is followed by the residue number and the new amino acid as in H134Q; DNase I variants with multiple mutation...

Evaluation of all non‐synonymous single nucleotide polymorphisms (SNPs) in the genes encoding human deoxyribonuclease I and I‐like 3 as a functional SNP potentially implicated in autoimmunity

“…It seems plausible that the appearance of the new Cys residue distal from the essential Cys residues at the C-terminal end of the protein might not affect formation of the normal disulfide bond. Pan et al [35] have proposed that the amino acid residues in the DNase I protein be classified into four groups based on their different function roles; the first, second, third and fourth classes are composed of the four residues absolutely essential for catalysis, the three crucial acidic residues serving as ligands for metal ion chelation, the seven residues critically involved in DNA-interacting positions encircling the active site, and the 13 residues interacting with DNA distal from the active site, respectively. Among the non-synonymous SNPs in DNASE1 examined, the amino acid residues corresponding to p.Asp190His, and to p.Arg133Gln, p.Arg133Leu and p.Asn192Ile, belong to the second and the third classes, respectively.…”

Ca2+‐dependent activity of human DNase I and its hyperactive variants

“…Thus, the electrostatic interactions introduced in these variants have a variety of effects on DNase I activity. In addition to increasing the functional catalytic activity by binding to DNA more effectively and being more resistant to inhibition by salt than wild-type DNase I~Pan & Lazarus, 1997, 1998Pan et al, 1998aPan et al, , 1998b! the Class B hyperactive variants are also relatively independent of Ca 2ϩ for their activity.…”

“…The pharmacological and clinical importance of DNase I has led us to engineer hyperactive variants that have a different functional mechanism for DNA cleavage~Pan & Lazarus, 1997, 1998!, as well as actin-resistant variants of DNase I that are no longer inhibited by actin~Ulmer et al, 1996! ; characterization of these variants under biologically relevant ex vivo conditions has shown a marked improvement in potency compared to wild-type~Pan et al, 1998a Structural-functional analysis has implicated several critical DNAcontacting residues surrounding the active site and other DNAinteracting positions that are distal to the active site and contribute less to activity~Jones et al, 1996;Pan et al, 1998b;Evans et al, 1999!. A selected group of amino acids at the DNA binding interface, yet distal to the active site, has been systematically replaced with positively charged residues, yielding hyperactive human DNase I variants that are substantially more active than wild-type under physiological saline conditions~Pan & Lazarus, 1997Lazarus, , 1998 In the present work, we investigated the effect of the Ca 2ϩ ions on the DNA cleavage activity of wild-type human DNase I and its hyperactive variants. Furthermore, we determined the role of the two structural Ca 2ϩ binding sites through site-directed mutagenesis.…”

Engineering a waste management enzyme to overcome cancer resistance to apoptosis: adding DNase1 to the anti-cancer toolbox

“…This substitution has been reported to decrease WT DNase1 activity by B10-fold. 47 We found that knocking down the activity site resulted in restoration of cell survival ( Figure 5), as well as reduction in the number of apoptotic cells (Figure 8) and chromatin-degrading effect (Figure 7). These findings suggest that C11 and C13 triggered apoptosis by direct cleavage of the genomic DNA through their catalytic site.…”