Evidence that a catalytic glutamate and an ‘Arginine Toggle’ act in concert to mediate ATP hydrolysis and mechanochemical coupling in a viral DNA packaging motor

Abstract

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.

Atomistic Basis of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor

“…This rotation could either actively pull the trans-acting loops of the nieghboring subunit with it, or passively move these loops by relieving the tension imposed by the lid subdomain in the ATP-bound configuration. This agrees well with prior structual, biochemical, and simulation data that suggest both ATP binding and nucleotide release actuate rotation of the lid subdomain (Hilbert et al, 2015;Ortiz et al, 2019).…”

“…These differences may be due to subtle differences in the way ATP is bound; subunits wherein adenosine binds the Q-motif have lid subdomain motion projected towards the ATPase binding site, whereas subunits wherein adenosine is not bound to the Q-motif do not. Although prior predictions suggested that the apoto-ATP-bound transition is characterized by an "open-to-closed" transition (Ortiz et al, 2019), our simulations suggest this transition could also be characterized as a "flexible-to-rigid" state transition.…”

“…Our model ascribes the ATP-binding induced lid subdomain rotation (Fig. 5), also observed in (Hilbert et al, 2015;Ortiz et al, 2019), as the force-generating conformational change. Importantly, phosphate release in one subunit releases that subunit's grip on DNA, and coordinates translocation of the neighboring DNA-bound subunit.…”

Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation

“…We note that the Walker A arginine residue moves into the binding pocket upon ATP-binding and moves out of the binding pocket in the absence of the γ-phosphate. We have previously reported that this conserved Walker A arginine in phage λ functions analogously to the “sensor II motif” arginine found in AAA+ enzymes, whose role is to sense the presence of the γ-phosphate and induce further conformational changes (10). The results presented here further support this assignment.…”

“…A common feature among ASCE enzymes is the presence of a P-loop/Walker A (WA) and Walker B (WB) motifs which bind ATP and catalyze hydrolysis, respectively (8, 9). The feature that distinguishes ASCE enzymes from other superfamilies of ATPases is the presence of a conserved glutamate residue downstream of the WB motif that is necessary for catalytic activity (6, 10, 11). Like most ASCE systems, viral packaging ATPases assemble as oligomeric rings that thread their biopolymer substrate through their central pores (12, 13).…”