| dc.description.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.
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