Strong subunit coordination drives a powerful viral DNA packaging motor

Abstract

Terminase enzymes are viral motors that package DNA into a preformed capsid and are of interest both therapeutically and as potential nano-machines. The enzymes excise a single genome from a concatemeric precursor (genome maturation) and then package the duplex to near-crystalline density (genome packaging). The functional motors are oligomers of protomeric subunits and are the most powerful motors currently known. Here, we present mechanistic studies on the terminase motor from bacteriophage λ. We identify a mutant (K76R) that is specifically deficient in packaging activity. Biochemical analysis of this enzyme provides insight into the linkage between ATP hydrolysis and motor translocation. We further use this mutant to assemble chimeric motors with WT enzyme and characterize the catalytic activity of the complexes. The data demonstrate that strong coordination between the motor protomers is required for DNA packaging and that incorporation of even a single mutant protomer poisons motor activity. Significant coordination is similarly observed in the genome maturation reaction; however, although the motor is composed of a symmetric tetramer of protomers, the maturation complex is better described as a "dimer-of-dimers" with half-site reactivity. We describe a model for how the motor alternates between a stable genome maturation complex and a dynamic genome packaging complex. The fundamental features of coordinated ATP hydrolysis, DNA movement, and tight association between the motor and the duplex during translocation are recapitulated in all of the viral motors. This work is thus of relevance to all terminase enzymes, both prokaryotic and eukaryotic.

Fig. 1.

Maturation and packaging of a λ genome by the terminase enzyme. Details are provided in the main text.

Fig. 2.

The gpA-K76R mutation specifically abrogates DNA packaging activity. Sedimentation distribution profiles for the purified protomers in low salt buffer (A) and the assembled tetramer species in high salt buffer (B). WT, K76R, and a 50:50 mix of the two protomers are shown in blue, red, and violet, respectively. (C) Catalytic activity of the WT (blue) and K76R (red) terminase protomers. Error bars represent SD (n = 3).

Fig. 3.

The DNA packaging motor shows strong coordination between the protomers. (A) Relative packaging activity as a function of f is indicated in black squares. Error bars represent SD (n = 3). The predicted ensemble activity of the motors in solution was calculated from Eq. 3 incorporating zero (blue line; SSE = 0.91), 100% (red line; SSE = 4.55 × 10⁻³), 90% (red dashed line; SSE = 1.98 x10⁻²), and 80% (red dotted line; SSE = 5.10 × 10⁻²) coordination in the individual motors (Fig. S2B). (B) The experimental data from A is redisplayed, and the predicted ensemble activity of motors containing three (purple; SSE = 5.65 × 10⁻²), four (red; SSE = 4.55 × 10⁻³), five (blue; SSE = 6.49 × 10⁻³), and six (green; SSE = 3.03 × 10⁻²) protomers was calculated from Eq. 3 assuming 100% coordination.

Fig. 4.

The genome maturation complex shows complex coordination. Relative strand separation activity as a function of f is indicated in black diamonds. Error bars represent SD (n = 3). The predicted ensemble activity of a symmetric tetrameric complex in solution was calculated from Eq. 3 assuming zero (blue line; SSE = 2.84 × 10⁻²), 50% (purple line; SSE = 2.99 × 10⁻³), or 100% (red line; SSE = 2.51 × 10⁻²) coordination in the individual motors (Fig. S3). The predicted ensemble activities of a simple dimer model (black dots; SSE = 5.71 × 10⁻⁴) and a dimer of dimers model (green line; SSE = 5.65 × 10⁻⁴) are shown assuming 100% coordination (Figs. S4 and S5B).