Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein.
Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.
Thomsen
Malene S
MS
Institute of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Franssen
Lars
L
Launholt
Dorte
D
Fojan
Peter
P
Grasser
Klaus D
KD
D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024361:Amino Acids, Basic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047390:Casein Kinase II; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D024243:HMGB1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D003313:Zea mays / Q000378:metabolism*
D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D024243:HMGB1 Protein; D011994:Recombinant Proteins; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D005022:Ethyldimethylaminopropyl Carbodiimide