Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein.
The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.
Das
Aditi
A
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1079, USA.
Rajagopalan
Lavanya
L
Mathura
Venkatarajan S
VS
Rigby
Samuel J
SJ
Mitra
Sankar
S
Hazra
Tapas K
TK
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D045647:DNA Glycosylases / Q000737:chemistry* / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers
D010455:Peptides; C045975:5-hydroxyuracil; D006639:Histidine; D014498:Uracil; D004247:DNA; D001120:Arginine; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C473642:NEIL2 protein, human; D015032:Zinc; D003545:Cysteine; D010100:Oxygen