RNA Packaging Motor: From Structure to Quantum Mechanical Modelling and Sequential‐Stochastic Mechanism

Abstract

The bacteriophages of the Cystoviridae family package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate, i.e. the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work.

RNA remodeling by hexameric RNA helicases

“…50 Thus, while there are many structural features shared between ϕ12 P4 and ec Rho, it is clear that their ATP hydrolysis and RNA translocation mechanisms are not entirely alike (see also preceding section on NTP hydrolysis). Accordingly, the mechanism that has been put forward from both kinetic testing 31,56 and the ϕ12 P4 structures (Fig. 1E) 54,55 differs from those proposed for ec Rho.…”

“…31,54 The sequential mechanism of ϕ12 P4 may involve intersubunit cooperation in subsets of three to four neighboring subunits (see also below). 31,55,56 While ec Rho and P4 share many features of sequential mechanism, there are clear differences between their respective catalytic cycles. The Rho hexamer exhibits cooperative and tight (K d ~ 1 μM) ATP binding 52,57 mediated by subunit closure and opening of catalytic sites at the subunit interfaces.…”

“…54,55 The model was further supported by ATPase kinetics obtained with homogeneous P4 rings 31 and with reconstituted hexamers containing wild-type and mutant subunits. 56 In the structures, the ϕ12 P4 hexamer contains a central channel that is comparable to that of Rho. 55 The channel is occupied by the L1 (which is disordered in all structures and was only modeled in some) and L2 loops as well as by the N-terminal sections of helices α6 (Fig.…”

“…This mechanism posits that only three to four ϕ12 P4 subunits bind RNA at any time (Fig. 5C) 56 although all active sites sequentially go through a [T→T→T→T→T*→D] cycle (Fig. 1E) and effect translocation in net steps of ~2 nt per molecule of ATP hydrolyzed.…”

Mechanism of RNA Packaging Motor

“…Computed data for the three and four cooperating subunit model are shown as solid and dotted curves , respectively. Adapted from Telenius et al ( 2008 ) In contrast to P4, cooperativity in other hexameric helicases, such as Rho and T7 gp4, is achieved by cooperative ATP binding in which the ATP affi nity ( K d in the lower m M range) is modulated by RNA binding. Despite this, a sequential mechanism of hydrolysis similar to the one proposed for P4 has been put forward for these two helicases (Liao et al 2005 ;Adelman et al 2006 ) .…”