Structural Basis of Mechanochemical Coupling in a Hexameric Molecular Motor

Abstract

The P4 protein of bacteriophage 12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small ϳ1 Å changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNAbinding site that moves along the mechanical reaction coordinate. The lever is flanked by ␥-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.Helicases are molecular motors that unwind doublestranded nucleic acids using the energy of NTP hydrolysis. Helicases have been divided into four superfamilies (SF1-SF4) 8 based on the sequence identity among the conserved helicase motifs (1). Helicases belonging to SF1 and SF2 (e.g. RecBCD and RecQ) function as monomers and provide simple model systems for studying DNA translocation and strand separation. An inchworming mechanism of translocation and unwinding was proposed on the basis of extensive structural and biochemical data (2, 3).Members of the SF3 and SF4 function as hexameric rings that encircle their polynucleotide substrates (DNA or RNA) and translocate along one strand. A recent landmark structural study on human papilloma virus helicase E1 (SF3 helicase) demonstrated that DNA is bound to conserved ␤-hairpins on multiple E1 subunits (4). A correlation between the positions of E1 ␤-hairpin and the conformations of the corresponding ATPbinding site provided evidence for sequential hydrolysis and translocation by an "escort" mechanism (4). Another important study on Rho transcription terminator (SF5 helicase (5)) has shown that RNA binds to multiple sites (Q loop, R loop, and P loop) in a cleft between two neighboring Rho protomers (6). Rho helicase also utilizes a sequential hydrolysis scheme (7); however, the translocation along RNA was proposed to proceed by a directed hand-off mechanism mediated by local conformational changes within the Q loops. A well known SF4 helicase, gp4 from bacteriophage T7 (T7 gp4), also employs a sequential ATP hydrolysis (8 -12). A set of gp4 x-ray structures suggested that ATP hydrolysis triggers sequential subunit rotations, which were proposed to effect nucleic acid...

On helicases and other motor proteins

“…A concerted hydrolysis mechanism has been described for Tag, but because of the many sequence, structural, and functional similarities, we expect that all SF3 helicases, including Tag, to coordinate DNA and operate by a sequential hydrolysis escort mechanism described above for E1. A sequential hydrolysis mechanism with coordination among the subunits has also been proposed for the f12 dsRNA packaging motor P4 [80,81,82 ]. In the case of T7gp4, DNAdependent ATPase activity has been shown to require active ATPase sites at all six subunit interfaces, inconsistent with a probabilistic hydrolysis model [66 ].…”

RNA remodeling by hexameric RNA helicases

“…5A). 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.…”

“…3A). 11,15 Accordingly, crystal structures obtained for ec Rho 39,40,50,66 and ϕ12 P4, 54,55 indicate that the two enzymes have similarly folded central domains (128 residues superpose with an rms deviation of 2.8 Å) 54 with conserved motifs at comparable locations (Fig. 3B).…”

“…1E). 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.…”

“…This was done by immobilizing the lever (see description of P4 structure, below), i.e., by stopping movement along the mechanical reaction coordinate, 54 which completely abolished the basal ATPase activity. The conclusion protein P1 via their C-terminal facets and thus provide directionality for packaging.…”