Mapping the DNA- and zinc-binding domains of ASR1 (abscisic acid stress ripening), an abiotic-stress regulated plant specific protein.
Abscisic acid stress ripening (ASR1) is a highly charged low molecular weight plant specific protein that is regulated by salt- and water-stresses. The protein possesses a zinc-dependent DNA-binding activity (Kalifa et al., Biochem. J. 381 (2004) 373) and overexpression in transgenic plants results in an increased salt-tolerance (Kalifa et al., Plant Cell Environ. 27 (2004) 1459). There are no structure homologs of ASR1, thus the structural and functional domains of the protein cannot be predicted. Here, we map the protein domains involved in the binding of Zn(2+) and DNA. Using mild acid hydrolysis, and a series of ASR1 carboxy-terminal truncations we show that the zinc-dependent DNA-binding could be mapped to the central/carboxy-terminal domain. In addition, using MALDI-TOF-MS with a non-acidic matrix, we show that two zinc ions are bound to the amino-terminal domain. Other zinc ion(s) bind the DNA-binding domain. Binding of zinc to ASR1 induces conformational changes resulting in a decreased sensitivity to proteases.
Rom
Slava
S
Department of Life Sciences and Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel.
Gilad
Ayelet
A
Kalifa
Yossi
Y
Konrad
Zvia
Z
Karpasas
Mark M
MM
Goldgur
Yehuda
Y
Bar-Zvi
Dudy
D
D000040:Abscisic Acid; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018551:Solanum lycopersicum; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000378:metabolism*
C091853:Asr1 protein, Lycopersicon esculentum; D004268:DNA-Binding Proteins; D010940:Plant Proteins; D000040:Abscisic Acid; D004247:DNA; D015032:Zinc