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J. Biol. Chem., Vol. 283, Issue 6, 3607-3617, February 8, 2008

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Structural Basis of Mechanochemical Coupling in a Hexameric Molecular Motor^*

Denis E. Kainov¹², Erika J. Mancini¹³, Jelena Telenius, Jií Lísal⁴, Jonathan M. Grimes⁵, Dennis H. Bamford, David I. Stuart⁶, and Roman Tuma⁷

From the Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Viikki Biocenter P. O. Box 65, Helsinki FIN-00014, Finland and Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN, United Kingdom

The P4 protein of bacteriophage 12 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 1 Å 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 -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.

Received for publication, August 1, 2007 , and in revised form, October 16, 2007.

The atomic coordinates and structure factors (code 2VHC, 2VHJ, 2VHQ, 2VHT, 2VHU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Human Frontiers Science Programme, the UK Medical Council, and the Academy of Finland ("Finnish Centre of Excellence in Virus Research 2006-2011") Grants 1206926 (to R. T.), 1202855, and 1202108 (to D. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Both authors contributed equally to this work.

² Fellow of the Finnish National Graduate School in Informational and Structural Biology. Present address: Laboratoire National de Santé, 20A, rue Auguste Lumiere, L-1950 Luxembourg.

³ Supported by EMBO Postdoctoral Fellowship ALTF-192 and is now supported by the UK Royal Society.

⁴ Supported by the Viikki Graduate School in Biosciences. Present address: Stanford University School of Medicine, Dept. of Molecular and Cellular Physiology, Beckman Center B151, 279 Campus Dr., Stanford, CA 94305-5345.

⁵ Supported by the UK Royal Society.

⁶ Supported by the UK Medical Research Council. To whom correspondence may be addressed: Tel.: 44-1865287567; Fax: 44-1865287547; E-mail: dave{at}strubi.ox.ac.uk.

⁷ To whom correspondence should be addressed: Astbury Centre for Structural Molecular Biology and Institute of Cellular and Molecular Biology, University of Leeds, LS2 9JT, United Kingdom. Tel.: 44-1133433080; Fax: 44-1133437897; E-mail: r.tuma{at}leeds.ac.uk.

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