Structure and mechanism of the RuvB Holliday junction branch migration motor

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.     

Cooperative mechanism of RNA packaging motor

P4 is a hexameric ATPase that serves as the RNA packaging motor in double-stranded RNA bacteriophages from the Cystoviridae family. P4 shares sequence and structural similarities with hexameric helicases. A structure-based mechanism for mechano-chemical coupling has recently been proposed for P4 from bacteriophage phi12. However, coordination of ATP hydrolysis among the subunits and coupling with RNA translocation remains elusive. Here we present detailed kinetic study of nucleotide binding, hydrolysis, and product release by phi12 P4 in the presence of different RNA and DNA substrates. Whereas binding affinities for ATP and ADP are not affected by RNA binding, the hydrolysis step is accelerated and the apparent cooperativity is increased. No nucleotide binding cooperativity is observed. We propose a stochastic-sequential cooperativity model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. The translocation step appears coupled to hydrolysis, which is coordinated among three neighboring subunits. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive.     

Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility

PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.