ABSTRACT
The WRKY proteins comprise a major family of transcription factors that are essential in pathogen and salicylic acid responses of higher plants as well as a variety of plant-specific reactions. They share a DNA binding domain, designated as the WRKY domain, which contains an invariant WRKYGQK sequence and a CX4-5CX22-23HXH zinc binding motif. Herein, we report the NMR solution structure of the C-terminal WRKY domain of the Arabidopsis thaliana WRKY4 protein. The structure consists of a four-stranded beta-sheet, with a zinc binding pocket formed by the conserved Cys/His residues located at one end of the beta-sheet, revealing a novel zinc and DNA binding structure. The WRKYGQK residues correspond to the most N-terminal beta-strand, kinked in the middle of the sequence by the Gly residue, which enables extensive hydrophobic interactions involving the Trp residue and contributes to the structural stability of the beta-sheet. Based on a profile of NMR chemical shift perturbations, we propose that the same strand enters the DNA groove and forms contacts with the DNA bases.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/metabolism , DNA-Binding Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Base Sequence , Binding Sites , DNA, Plant/genetics , DNA, Plant/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Solutions , Static Electricity , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The B3 DNA binding domain is shared amongst various plant-specific transcription factors, including factors involved in auxin-regulated and abscisic acid-regulated transcription. Herein, we report the NMR solution structure of the B3 domain of the Arabidopsis thaliana cold-responsive transcription factor RAV1. The structure consists of a seven-stranded open beta-barrel and two alpha-helices located at the ends of the barrel and is significantly similar to the structure of the noncatalytic DNA binding domain of the restriction enzyme EcoRII. An NMR titration experiment revealed a DNA recognition interface that enabled us to propose a structural model of the protein-DNA complex. The locations of the DNA-contacting residues are also likely to be similar to those of the EcoRII DNA binding domain.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Acclimatization/physiology , Amino Acid Sequence/physiology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Binding Sites/genetics , Cold Temperature , DNA-Binding Proteins/genetics , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Evolution, Molecular , Models, Molecular , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular , Phylogeny , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/genetics , Sequence Homology, Amino AcidABSTRACT
Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.
