The nuts and bolts of ring-translocase structure and mechanism

Abstract

Ring-shaped, oligomeric translocases are multisubunit enzymes that couple the hydrolysis of Nucleoside TriPhosphates (NTPs) to directed movement along extended biopolymer substrates. These motors help unwind nucleic acid duplexes, unfold protein chains, and shepherd nucleic acids between cellular and/or viral compartments. Substrates are translocated through a central pore formed by a circular array of catalytic subunits. Cycles of nucleotide binding, hydrolysis, and product release help reposition translocation loops in the pore to direct movement. How NTP turnover allosterically induces these conformational changes, and the extent of mechanistic divergence between motor families, remain outstanding problems. This review examines the current models for ring-translocase function and highlights the fundamental gaps remaining in our understanding of these molecular machines.

Figure 1

RecA and AAA+ ring translocases [1]. (a) Phylogenetic tree showing several RecA and AAA+ clades of the ASCE superfamily. Representative family members are shown, with polymer-translocases highlighted red. At center is a diagram of the conserved ASCE fold highlighting key elements of the NTPase active site. Abbreviations: WA – Walker A, WB – Walker B, CE – catalytic glutamate, RF – arginine finger, SF1-6 – helicase superfamilies 1 through 6, PS2 – pre-sensor II insert, H2I – helix 2 insert, HCLR – (HslU, ClpABC-CTD, LonAB, RuvB), ABC – ATP Binding Cassette, AAA+ - ATPases Associated with diverse cellular Activities, ASCE – Additional Strand, Catalytic glutamate ([1]). (b) Substrate-bound Rho (PDB ID: 3ICE) and E1 (PDB ID: 2GXA) hexamers shown as RecA and AAA+ exemplars, respectively. The ATPase status of each subunit is labeled; loops in the translocation pore and nucleotide molecules are differentially colored accordingly (ATP-type – blue, ADP-type – magenta, nucleotide exchange – green); arginine fingers are shown as cyan sticks.

Figure 2

Structural comparisons of RecA and AAA+ ring translocases. (a) Cartoon representation of AAA+ (E1) and RecA-type (Rho) ASCE domains. Conserved α-helices and β-strands are colored pale blue and yellow, respectively. Nucleotide-binding elements are green, substrate-binding loops magenta, and non-conserved insertions grey. Nucleotides and the Arg-finger are shown as ball-and-stick. (b,c) Close-up of the E1 (panel b) and Rho (panel c) active sites. The NTP-binding subunit is green, and the subunit donating the Arg-finger is cyan. Conserved active site elements, nucleotide, active site water molecules and ions are shown (a Cl⁻ ion (E1) or BeF₃ species (Rho) occupies the position of the γ-PO₄ moiety). (d) Superposition of a single ASCE domain within the Rho (beige) and E1 (cyan) hexamers (inset) highlights the orientational offset between the RecA and AAA+ translocation pores (magenta).

Figure 3

NTP hydrolysis models for ring-translocases. NTPase states are labeled as follows: T (blue) – NTP state, DP (purple) – NDP + P_i state, E (green) – nucleotide exchange state. (a) Symmetric rotary mechanism. (b) Asymmetric rotary mechanism. (c) Concerted mechanism. (d) Stochastic mechanism. See text for details.

Figure 4

Molecular determinants of translocation polarity. (a) Rho and E1 pore loops bound to ssRNA and ssDNA, respectively (5’ end at top). Loops are labeled colored by subunit nucleotide state in accord with Fig 1b. (b) A “nut-and-bolt” schema for considering translocation polarity. The direction of movement of the nut on the leftmost bolt can be reversed by: 1) reversing the substrate binding orientation (flipping the bolt, middle left panel), 2) reversing the chirality of the pore loop “staircase” (inverting the threads on the nut, middle right panel), or 3) reversing the NTPase cycle progression (turning the nut in the opposite direction, rightmost panel). (c) Schematic of Rho and E1 (hexamer 1) as viewed from the 5’ end of their substrates, illustrating their respective NTP hydrolysis polarities. The subunit labeled (T*?) in E1 reflects the adoption of ADP or ATP-like states by this protomer in different hexamers. Subunits are colored according to nucleotide state as in Fig 1b. Nucleic acid is shown as a spiral of grey circles.