We have recently become interested in how viruses package their genomes into capsids. In particular, many DNA viruses utilize a powerful molecular motor during assembly to translocate DNA into a preformed capsid shell. Our collaborator, Dr. Douglas Smith, has used elegant single-molecule experiments with optical traps (Fig. 7) to show that these motors are capable of generating forces in excess of 60 pN and packaging DNA at rates of 200 to 2000 bp/s, making them the most powerful molecular motor known to mankind. The molecular mechanisms by which these motors generate such large forces and high packaging speeds remain largely unknown. Our lab is using a range of computational tools, in combination with experiments in the Smith lab, to resolve these mechanisms. Uncovering such mechanisms would not resolve a fundamental problem in virology but also provide insights into combating viral infections like herpes and designing synthetic mimics of these powerful molecular motors.
We started by investigating the DNA packaging mechanism of the T4 bacteriophage motor by carrying out single-molecule DNA packaging measurements and free energy calculations via the MM-GBSA approach. In particular, we tested a previously proposed mechanism of packaging in which the T4 motor protein translocates DNA by transitioning between an extended and a compact state due to electrostatic interactions between complimentarily charged residues across an interface between two domains of the motor (Fig. 7). We showed that site-directed alterations in these residues cause force dependent impairments of motor function that correlate well with computed changes in free-energy differences between the two states, thus providing support for the proposed model. We also proposed an energy landscape model of motor activity under external loads that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle (Fig. 8).
In a subsequent study, we carried out free energy decomposition analysis to identify key molecular interactions and residues involved in force generation (Fig. 9). We found that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identified specific charged and uncharged residues, and the specific interactions that these residues mediate, at the interface that play a dominant role in the compaction transition. The computed contributions were found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations. We are currently examining other aspects of packaging, including the 3D arrangement of the subunits comprising the T4 motor and the conformational relaxation of DNA within the capsids.