ABSTRACT
The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222(1) and P4(1)2(1)2, at a resolution of 1.28 and 3.05 A, respectively. tPphA belongs to a large and widely distributed subfamily of Mg(2+)/Mn(2+)-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.
Subject(s)
Cyanobacteria/enzymology , Phosphoprotein Phosphatases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Catalysis , Cations, Divalent/metabolism , Crystallography, X-Ray , DNA Mutational Analysis , Escherichia coli/genetics , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrolysis , Isoenzymes , Kinetics , Manganese/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Phosphoprotein Phosphatases/analysis , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/isolation & purification , Phosphoprotein Phosphatases/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Solubility , Substrate SpecificityABSTRACT
The chromatin accessibility complex (CHRAC) is an abundant, evolutionarily conserved nucleosome remodeling machinery able to catalyze histone octamer sliding on DNA. CHRAC differs from the related ACF complex by the presence of two subunits with molecular masses of 14 and 16 kDa, whose structure and function were not known. We determined the structure of Drosophila melanogaster CHRAC14-CHRAC16 by X-ray crystallography at 2.4-angstroms resolution and found that they dimerize via a variant histone fold in a typical handshake structure. In further analogy to histones, CHRAC14-16 contain unstructured N- and C-terminal tail domains that protrude from the handshake structure. A dimer of CHRAC14-16 can associate with the N terminus of ACF1, thereby completing CHRAC. Low-affinity interactions of CHRAC14-16 with DNA significantly improve the efficiency of nucleosome mobilization by limiting amounts of ACF. Deletion of the negatively charged C terminus of CHRAC16 enhances DNA binding 25-fold but leads to inhibition of nucleosome sliding, in striking analogy to the effect of the DNA chaperone HMGB1 on nucleosome sliding. The presence of a surface compatible with DNA interaction and the geometry of an H2A-H2B heterodimer may provide a transient acceptor site for DNA dislocated from the histone surface and therefore facilitate the nucleosome remodeling process.
Subject(s)
DNA/chemistry , Drosophila Proteins/chemistry , Histones/chemistry , Nucleoproteins/chemistry , Nucleosomes/metabolism , Adenosine Triphosphatases/chemistry , Amino Acid Sequence , Animals , Catalysis , Cloning, Molecular , Crystallography, X-Ray , Dimerization , Escherichia coli/metabolism , Gene Deletion , Glutathione Transferase/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Nucleosomes/chemistry , Protein Binding , Protein Biosynthesis , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Transcription Factors/chemistry , XenopusABSTRACT
Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.
