Atomic Basis of Coordination, Force Generation, and Translocation in Ring ATPases

Abstract

<p>Many vital biological tasks, such as protein degradation, DNA strand separation, and viral DNA packaging are performed by ring NTPase assemblies. These assemblies harvest energy from NTP binding and hydrolysis in order to translocate their biopolymer substrate through their central pores. Single-molecule characterization demonstrated that these assemblies are highly coordinated and produce forces an order of magnitude larger than most molecular motors. Recently, many structures of these assemblies have been experimentally solved and resulting globular translocation models have been proposed. While these static structures have provided great insights into how molecular motors assemble, the specific molecular mechanisms that promote, regulate, and coordinate the dynamic translocation processes remain poorly understood. In this dissertation, I use computational tools to model ring ATPase molecular motors in order to elucidate such mechanisms. Initially, I focus on viral packaging ATPases and then generalize my findings to a broader class of motors by studying FtsK-like and AAA+ motors. For all systems, atomistic molecular dynamics simulations were used to calculate free-energy landscapes that predict conformational changes, predict mutual-information-based signaling pathways that couple enzymatic and mechanical activities, predict principal components of motion that describe the enzyme’s native function, and predict the effects of mutagenesis in silico. For viral packaging ATPases, I first predicted that a strictly conserved Walker A arginine residue functions analogously to a sensor II motif arginine found in AAA+ systems, and that it is used to couple ATP binding to lid subdomain rotation. Second, I predicted how mutations in the Walker A and Walker B motifs could abrogate enzyme function. All these predictions were corroborated by collaborators’ extensive experimental characterization. Third, I helped build the first structure of an actively packaging viral ATPase motor into the cryo-EM reconstruction and led the biological interpretation of the resulting structure. Fourth, I used molecular dynamics simulations of pentameric ATPase assemblies to predict how the assemblies respond to nucleotide-occupancy and presence of double-stranded DNA substrate. Based on the structure and simulations, I proposed the helical-to-planar model of viral DNA packaging, which is the first atomistic model that can predict the salient features of viral DNA packaging. Further, this model lays the groundwork of future work by predicting specific conformational changes and interactions that were otherwise obscure from experimental studies. Fifth, I tested a key proposal in my helical-to-planar model by using molecular dynamics simulations to investigate how nucleotide binding is coupled to substrate gripping. The resulting glutamate switch signaling pathway was corroborated by structural data and functional mutagenesis assays. Lastly, I investigated FtsK-like and AAA+ enzymes to probe for molecular mechanisms common to a broad class of translocating ring ATPases. From these studies, I identified a core set of principles that can be modularly added together to describe a number of different translocation models. In summary, the results presented in this dissertation describe fundamental mechanisms of translocating ring ATPase motors. When possible, my computational predictions were corroborated by experimental characterization. When experimental characterization was not yet possible, my predictions and derived models serve as a guide for future studies. The models I derived provide the first comprehensive description of the coordinated conformational changes that drive viral DNA packaging. Further, they have the potential to inform rational design of synthetic molecular motors and anti-viral therapeutics that target the genome packaging step. </p>