Structural basis of mechanochemical coupling in a hexameric molecular motor.
ABSTRACT The P4 protein of bacteriophage phi12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small approximately 1 A changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNA-binding site that moves along the mechanical reaction coordinate. The lever is flanked by gamma-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.
Article: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.[show abstract] [hide abstract]
ABSTRACT: Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.Nucleic Acids Research 11/2008; 36(20):6620-32. · 8.03 Impact Factor
Dataset: Yu 2010 Phi29[show abstract] [hide abstract]
ABSTRACT: Full text on "Mechanochemistry of a viral DNA packaging motor"
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ABSTRACT: The unwinding of RNA helices and the disruption of RNA-protein complexes are critical steps of cellular metabolism that are carried out by ubiquitous NTP-dependent enzymes named RNA helicases. Here, we review the structures, mechanisms, and biochemical properties of two RNA helicases known to adopt a homo-hexameric ring architecture: the P4 packaging motor of bacteriophage φ8, a Super-Family 4 helicase, and Escherichia coli's transcription termination factor Rho from the Super-Family 5 of helicases. We emphasize the many similarities as well as key differences that characterize the Rho and P4 motor mechanisms and highlight important questions that remain to be addressed.RNA biology 11/2010; 7(6):655-66. · 5.56 Impact Factor