During assembly of many viruses a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A “P-loop” motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectible levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectible yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type, but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A&R, while G81A and S83A exhibit strong impairments consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.
We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.
Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.Viral DNA packaging motors are remarkable biological nanomachines that are capable of packaging chromosomes to near-crystalline density into viral capsids against enormous internal forces (1, 2). Such motors are present in many doublestranded DNA bacteriophages (3), the medically relevant herpesviruses (4), and possibly in poxviruses and adenoviruses (5). The well characterized genetics and biochemistry of the bacteriophage system make it an ideal model system for investigating the structure-function relationships of DNA-packaging machines (6, 7). The "terminase enzyme" is the major active component of the packaging motor responsible for DNA translocation. It is composed of multiple heterotrimer units, each containing a large gpA subunit and two small gpNu1 subunits (8). This ATP-powered complex mediates a number of different activities needed to package unit length genomes from concatemeric substrates; these include endonuclease, strand separation, ATP hydrolysis, and DNA translocation catalytic activities. The terminase enzyme initiates packaging by binding to a cos site in a linear DNA concatemer composed of several viral genomes. Terminase introduces staggered nicks at the downstream cosN site, 12-bp apart to form the left mature genome end. The motor then binds to a procapsid and translocates DNA into the capsid interior. Packaging is terminated when the terminase encounters the next downstream cos site, which signals the right end of the genome; terminase again nicks the duplex, releasing the DNA-filled capsid. Recent evidence indicates that gpA 1 (gpNu1) 2 heterotrimers assemble into a tetrameric ring, which we presume is representative of the DNA maturation and packaging motor complex (9).Our earlier studies of the ATPase center of the packaging motor includ...
Optical tweezers facilitate measurement of piconewton-level forces and nanometer-level displacements and have broad applications in biophysics and soft matter physics research. We have shown previously that DNA molecules can be used as metrology standards to define such measurements. Force-extension measurements on two DNA molecules of different lengths can be used to determine four necessary measurement parameters. Here, we show that the accuracy of determining these parameters can be improved by more than 7-fold by incorporating measurements of the DNA overstretching transition and using a multi-step data analysis procedure. This method results in very robust and precise fitting of DNA force-extension measurements to the worm-like chain model. We verify the accuracy through independent measurements of DNA stretching, DNA unzipping, and microsphere contact forces.
ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine ‘toggles’ between interacting with a glutamate residue in the ‘lid’ subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an ‘open’ state to a ‘closed’ state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.
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