DNA recognition for virus assembly through multiple sequence-independent interactions with a helix-turn-helix motif

Abstract

The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses.

Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism

“…This assumption is supported by two observations: firstly, the large terminase nuclease domain resembles the RNase-H fold (14–19). Secondly, simultaneous binding of two metals, occupying positions A and B, has been observed in crystal structures of human cytomegalovirus (HCMV) UL89 nuclease and in the structure of the Sf6 gp2 nuclease in complex with β-thujaplicinol (14,18).…”

“…There is also an increased number of salt bridges (44): 9 versus 5 to 7 found in nucleases of mesophilic viruses (14–16,18,19,40). In addition, the β-hairpin, present only in viral nucleases, is more extended and ordered (Figure 1B).…”

“…Importantly, all of these flexible structural segments are conserved both in phage (14–16,40) and herpesvirus (18,19) terminases. We note that D347, implicated in facilitating metal binding at site B during the catalysis (see below), is located at the N-terminus of L 1 .…”

“…Recently, two different metal binding modes were reported for the Sf6 gp2 nuclease (14). In the first metal binding mode, the two Mg 2+ or Mn 2+ ions were modeled at ultra-short metal-metal distances of 2.42 and 2.64 Å, respectively, whereas in the second mode the two Mn 2+ ions are separated by 3.75 Å.…”

A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism

“…Motivated by the desire to see how such a small protein acts as a glycoside hydrolase, possibly reflecting unusual or unique strategies, we sought to determine the three-dimensional structure of Ss GH134. After removal of the MBP fusion partner, Ss GH134 crystals were obtained that diffracted to atomic resolution (approximately 1 Å), allowing the native structure to be solved by a novel “no-prior knowledge” molecular replacement method 23 using only an isolated standard helix as the search model (Tables 1 and S1). The Ss GH134 structure revealed a mixed α-helix/β-sheet fold with a strong superficial resemblance to family GH19 chitinases, GH22 C-type lysozymes, GH23 G-type lysozymes, and GH124 cellulases (Figures 2a, 3a–d, and S8).…”