Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes.
The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1-RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1-RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1-RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1-RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1-RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1-RuvBL2 complex does not fit the recent structural model of the yeast Rvb1-Rvb2 complex.
Niewiarowski
Andrew
A
Department of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK.
Bradley
Alison S
AS
Gor
Jayesh
J
McKay
Adam R
AR
Perkins
Stephen J
SJ
Tsaneva
Irina R
IR
D000074183:ATPases Associated with Diverse Cellular Activities; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D002352:Carrier Proteins; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486549:RUVBL1 protein, human; C122079:RUVBL2 protein, human