title: Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex.
abstract: TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, UK.
authors.forename: Teena
authors.initials: T
authors.lastname: Puri
authors.forename: Petra
authors.initials: P
authors.lastname: Wendler
authors.forename: Barbara
authors.initials: B
authors.lastname: Sigala
authors.forename: Helen
authors.initials: H
authors.lastname: Saibil
authors.forename: Irina R
authors.initials: IR
authors.lastname: Tsaneva
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003067:Coenzymes / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D008854:Microscopy, Electron; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D003067:Coenzymes; D009711:Nucleotides; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486549:RUVBL1 protein, human; C122079:RUVBL2 protein, human