An ancient anion-binding structural module in RNA and DNA helicases

Abstract

RNA and DNA helicases manipulate or translocate along single strands of nucleic acids by grasping them using a conserved structural motif. We have examined the available crystal structures of helicases of the two principal superfamilies, SF1 and SF2, and observed that the most conserved interactions with the nucleic acid occur between the phosphosugar backbone of a trinucleotide and the three strand-helix loops within a (beta-strand/alpha-helix)(3) structural module. At the first and third loops is a conserved hydrogen-bonded feature called a thr-motif, often seen at alpha-helical N-termini, with the threonine as the N-cap residue. These loops can be aligned with few insertions or deletions, and their main chain atoms are structurally congruent amongst the family members and between the two modules found as tandem pairs in all SF1 and SF2 proteins. The other highly conserved interactions with nucleic acid involve main chain NH groups, often at the helical N-termini, interacting with phosphate groups. We comment on how the sequence motifs that are commonly used to identify helicases map to locations on the module and discuss the implications of the conserved orientation of nucleic acid on the surface of the module for directional stepping along DNA or RNA.

Figure 1. Single-stranded nucleic acid binding (strand-helix)₃ modules.

(A) One portion, in blue, of the α/β sandwich helicase domain forms a ssRNA binding module; the nucleic acid binding loops are in green. The other portion, in black, is involved with binding ATP. SF1 and SF2 helicases have a tandem repeat of the domains D1 and D2. A single NTP molecule interacts with D1 and D2 in a “closed” configuration that brings the two single-stranded binding modules closer together. (B) Schematic topology of the module. Rectangular shapes represent strands of parallel β-sheet and cylinders represent α-helices. The ssRNA, seen behind the (strand-helix)₃ module, is shown in simplified representation as P = phosphate and R = ribose. The three strand-helix loops, in green, that bind the RNA are well conserved, whereas the two helix-strand loops, drawn thicker in blue, are less conserved. (C) Schematic representation of modules and conserved sequence motifs in SF1 and SF2 helicase domains. Sequence motifs Q, GG, and QxxR are characteristic of DEAD-box helicases and are absent in SF1 helicases. Interactions with RNA are indicated by black arrows. Interactions with ATP are indicated by dashed, gray arrows. Contacts represented by arrows are based on the crystal structure of the Vasa helicase. Modules M1 and M2 are indicated by blue and red and their nucleic acid binding loop sequences are green and magenta. The amino- and carboxy-termini are labelled N and C. (D) Drosophila melanogaster Vasa helicase structure with ssRNA. Modules M1 and M2 are shown in blue and red with the corresponding nucleic acid binding loops in green and magenta. RNA is yellow. This and other Figures of molecules were made using PyMOL (http://www.pymol.org).

Figure 2. The (strand-helix)₃ modules of the intron-exon junction component eIF4III (PDB code 2hyi).

(A) A tandem pair of modules bound to the ribose-phosphates of a pentanucleotide; in each module the first loop is blue, the second green and the third yellow. Phosphorus atoms are enlarged and orange. (B) A view of module M1 bound to the ribose-phosphates of a trinucleotide. (C) The mainchain (plus all threonine) atoms of the 3 M1 loops (using the same colors and in spacefill) bound to the ribose-phosphates of a trinucleotide. Interactions of module M1 loop 1 (D), loop 2 (E), and loop 3 (F) with the ribose-phosphates of RNA (main chain atoms only except for threonine and arginine). Figures are from different viewpoints.

Figure 3. Diagram of the three strand-helix loops of the (strand-helix)₃ modules of SF2 and SF1 helicases interacting with RNA and DNA trinucleotides.

(A) SF2 RNA helicases (B) SF2 DNA helicases (C) SF1 helicases. Hydrogen bonds are shown as dashed lines. The protein main chain is shown by ribbons and certain side chains are shown in grey. Pentagons and circles represent the alternating riboses or deoxyriboses and phosphates of the main chain of RNA or DNA. Residues of A are as for domain 1 of eIF4AIII. In B and C other interactions with the phosphates are present, but the diagram shows those hydrogen bonds in common with the RNA helicases.

Figure 4. Diagram of binding of modules M1 and M2 to nucleic acid.

A shows one arrangement commonly observed and B shows another. All helicases have one or the other arrangement but UvrD and RecD2 have A, the open conformation, in the absence of ATP, and B, the closed conformation, in its presence. These two conformations are thought to be important for the translocation of the helicase along ssDNA.

Figure 5. Overlay of helicase modules and nonhelicase proteins.

(A) Superposition of E coli DNA polymerase III (PDB code: 1em8, colored green) and module 2 of Vasa helicase (PDB code: 2db3, colored blue) shown from two different viewpoints. (B) Superposition of polynucleotide kinase (1ly1, colored red) and module 2 of archaeal helicase Hel308 (2p6r, colored blue) shown from two different viewpoints. Superpositions were performed using SSM, with the modules described in Table I. The superpositions with lowest RMSDs are presented.