LBNL Masthead A-Z IndexBerkeley Lab mastheadU.S. Department of Energy logoPhone BookJobsSearch
Rotary Firing in Ring-Shaped Protein Explains Unidirectionality Print
Wednesday, 28 April 2010 00:00

 

Hexameric motor proteins represent a complex class of molecular machines that variously push and pull on biological molecules using adenosine triphosphate (ATP) as chemical fuel. A specialized class of ring-shaped motor proteins, hexameric helicases, can unwind DNA strands and perform large-scale manipulations of single-stranded nucleic acids in processes such as DNA replication, DNA repair, and gene expression. To understand how certain hexameric helicases walk with directional polarity along single-stranded nucleic acids, Berkeley researchers used x-ray crystallography at the ALS to solve the structure of a hexameric helicase, the Rho transcription termination factor (from E. coli), bound to both ATP mimics and an RNA substrate. The results showed that Rho functions like a rotary engine: as the motor spins, it pulls RNA strands through its interior. Interestingly, the rotary firing order of the motor is biased so that the Rho protein can walk in only one direction along the RNA chain.

Rings Running in Reverse

The Rho factor is a ring-shaped motor protein made up of six subunits (or, in analogy to combustion engines, six "cylinders"). Such motor proteins (also known as hexameric helicases) are found in all organisms and are involved in unwinding and moving DNA and RNA strands (nucleic acids) around the cell. There are two subfamilies of hexameric helicases: AAA+ and RecA. Rho belongs to the RecA family, which is most common in bacteria. AAA+ motors are predominantly found in eukaryotes, including humans, as well as some human pathogens, such as the papillomavirus. Although these motors evolved from a common ancestor, they have distinct properties, most notably the predisposition to walk along nucleic acid tracks in opposite directions.

To understand how such a biological mechanism works and perhaps eventually develop a therapeutic drug that will gum up the works and stop the motor from doing its job, it helps to know how the protein is constructed. Thomsen et al. are the first group to determine the crystal structure of a RecA-class hexameric helicase in a translocation state bound to both its nucleic-acid track and a molecule that mimics the role of its chemical energy source, ATP. In doing so, they fortuitously caught an atomic-level snapshot of the motor in the act of tracking along an RNA chain. Their analysis showed that the proteins from different subfamilies move in opposite directions by reversing the rotational firing order of the ATP sites—essentially by reversing gears as opposed to turning around.

 

This movie shows a side view of the Rho protein motor in action. To clarify the mechanism, two of the six subunits have been removed. As ATP is released from binding sites, the subunits (green, purple, blue and gray) spiral around the RNA strand (orange), pulling the strand through the hole in the hexamer ring. This rotatory action enables the Rho motor to "walk" in one direction along the RNA strand.

Crystallization trials were conducted around a matrix of RNA substrates and nucleotide mimetics and initially resulted in three forms that diffracted to between 4 and 6 Å. Utilizing the capabilities of ALS Beamlines 12.3.1 and 8.3.1, the researchers were able to carefully screen hundreds of crystals, overcome a series of unique crystal defects, and collect a dataset diffracting to 2.8 Å. The structure is composed of six unique Rho protein chains, six ADP·BeF3 molecules, and six bases of an rU12 RNA substrate. The Rho model, which is remarkably asymmetric, provides six independent snapshots of the motor mechanism, revealing how ATP turnover is converted to mechanical energy that propels the protein along its RNA substrate.

Inspection of the model reveals that Rho binds to RNA in a helical conformation, using a "spiral staircase" arrangement of RNA binding loops that project from five of the protein subunits to interact with the RNA's sugar–phosphate backbone. The sixth subunit does not significantly contact the RNA and lies midway between the top and bottom steps of the staircase. The positional relationships between the six Rho subunits generate four distinct classes of ATP binding sites that together represent a complete ATP turnover cycle. The organization of these sites around the ring indicates that ATP is consumed by a sequential mechanism akin to a rotary or radial engine. The cycle of ATP binding, hydrolysis, and release is carried out as each subunit passes through the six conformational states observed in the structure, creating a rotary wave motion within the RNA binding loops to power nucleic acid translocation in the proper direction.

The structure of Rho bound to RNA and (ATP analogue) ADP·BeF3. a) Five of the six Rho subunits are illustrated as cartoons with a transparent surface (left). RNA (white) is visible in the center of the ring and ADP (cyan), BeF3 (black), and Mg2+ (yellow-green) are visible at the subunit interfaces. The expanded inset (right) shows the spiral staircase of protein loops (colored by subunit) that track the RNA (white) backbone. b) The four ATPase states in Rho are distinguished primarily by the distance between adjacent protein subunits, the positions of conserved catalytic residues (sidechains), and the presence of ordered water molecules (red spheres).

Comparison of Rho's structure to that of the remarkably similar human papillomavirus E1 protein indicates that Rho and E1 translocate in opposing directions because the radial sequence by which its multiple intermediate ATPase states activate to catalyze ATP turnover is inverted. This mechanism is analogous to reversing the firing order of cylinders in an internal combustion engine to reverse the direction of crankshaft rotation.

The position of ATPase intermediates (E, T, T*, and D) in Rho (left) and E1 (right) indicates that the ordinal direction of the ATPase cycle (large arrows) is inverted between the two enzymes. This property leads Rho and E1 to translocate in opposite directions along single-stranded nucleic acid substrates.

By providing the first high-resolution views of hexameric helicase translocation intermediates, the structures of Rho and E1 have broad implications for understanding motor mechanisms in general and can be used to guide further research on this class of proteins. Recent research on Rho and other systems such as the ClpX protein translocase, the bacteriophage φ29 packaging motor, minichromosome maintenance (MCM) protein, and the bacteriophage φ12 P4 packaging motor suggest that variations of the Rho and E1 mechanisms likely exist. Given their fundamental role in all known forms of cellular life, it is likely that modulating, reengineering, or inhibiting the functions of hexameric helicases will lead to exciting applications in basic research, biotechnology, and nanotechnology.

 


 

Research conducted by J.M. Berger (Univ. of California, Berkeley, and Berkeley Lab) and N.D. Thomsen (Univ. of California, Berkeley).

Research funding: National Institutes of Health and the G. Harold and Leila Y. Mathers Foundation. Operation of the ALS is supported by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences.

Publication about this research: N.D. Thomsen and J.M. Berger, "Running in reverse: The structural basis for translocation polarity in hexameric helicases," Cell 139, 523 (2009).

 

ALSNews Vol. 308