Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding

Abstract

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.

Keywords: C-terminal domain; DNA binding protein; DNA endonuclease; EcoR124; Escherichia coli (E. coli); GFP fusion; HsdR; X-ray crystallography; crystal structure; restriction-modification.

Figure 1.

Features of the C-terminal domain. A, crystal packing of the pHluorin-HsdR887 fusion protein with two pHluorin molecules (molecule A, brown; molecule B, blue) and the C-terminal domain of HsdR belonging to molecule A (red). Symmetric molecules are displayed in gray. B, overall structure of the C-terminal domain of HsdR consisting of α-helices 1–6 numbered from the N to the C terminus. C, crystal packing of the WT HsdR based on re-refinement of PDB entry 2W00 (molecule A, green; molecule B, blue; C-terminal domain, red). D, sample electron density of WT HsdR before (left panel) and after (right panel) re-refinement of the full structure with the C-terminal domain. The figure was prepared in PyMOL (A and C), YASARA (B), and Coot (D).

Figure 2.

A–C, full-length model of the HsdR subunit “front” (A) and “side” (B and C) views of the full-length HsdR model obtained by combining four previously known domains and the C-terminal domain. Domains are color-coded to match the first published HsdR structure (21); the C-terminal domain is shown in red. The catalytic triad (Asp¹⁵¹, Glu¹⁶⁵, and Lys¹⁶⁷) and ATP are marked in A. The corresponding electrostatic potential surfaces are shown on the right (blue, positive; red, negative). Positively charged surface areas in the C-terminal domain extend the proposed DNA path. The figure was prepared in YASARA.

Figure 3.

Interdomain interactions of the C-terminal domain. Shown is part of the HsdR subunit, showcasing the interface between the helicase 2 (magenta) and helical (green) and C-terminal domain (red). The molecular surface is drawn in transparent gray for each domain for atoms within the 5-Å distance from the interacting domain. The figure was prepared in YASARA.

Figure 4.

887 deletion mutant and WT HsdR binding to DNA in EMSA. The 10 nm oligonucleotide labeled with [γ³²P]ATP is in lane 1 (the corresponding band is marked free DNA), the oligonucleotide mixed with 40 nm MTase is in lane 2 (the band is marked M₂S-DNA), and either the C-terminal deletion mutant (lanes 3–6) or wild-type HsdR (lanes 7–10) was added to the DNA-MTase mixture at concentrations of 20, 40, 80, and 120 nm. Complexes with either one (R₁M₂S-DNA) or two (R₂M₂S-DNA) HsdR subunits are indicated with arrows where appropriate. Gel borders are outlined in gray.

Figure 5.

Occurrence of conserved regions in the C-terminal domain of putative HsdR sequences broken by sequence identity to R.EcoR124. Most of the sequences (∼92%) share 20–40% sequence identity with the HsdR of EcoR124, whereas ∼3% fall into 65–100%, representing the Type IC family. Four conserved regions were identified in sequences as remote as 40–50%.

Figure 6.

Multiple sequence alignment of the C-terminal domain of putative and described HsdR subunits. The alignment was performed for two biochemically described Type IC HsdR subunits (EcoR124 and NgoAV) and five putative proteins with a sequence identity of ∼44–71% compared with R.EcoR124. The sequences were retrieved from REBASE (1). The cladogram was produced using http://www.phylogeny.fr/ (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party–hosted site) (59) and PhyML (60); the alignment was performed in Clustal Omega (58). Only the C-terminal domain (amino acids 887–1038) is shown, although the alignments were performed with full-length subunits. The numbers on top of the sequence denote the EcoR124 numbering. Conserved residues are highlighted (⁸⁸⁷EXNXDYIL⁸⁹⁴ motif, blue; ⁹²⁵RXKXXLXXXFI⁹³⁵, orange; ⁹⁹⁶G—¹⁰⁰⁴PXXS¹⁰⁰⁷, green; ¹⁰¹⁶KKXXXXXK¹⁰²³, yellow; others, gray). The residues involved in inter- and intradomain contacts are marked with triangles colored according to the residue's contact partner (the domain coloring scheme is the same as in Fig. 2). The secondary structure elements are given according to the EcoR124 sequence (gray bars, α-helix numbered as in Fig. 1B; blue bars, 3₁₀-helix; green bars, turn).

Figure 7.

Motifs in the C-terminal domain of HsdR. A–D, conserved motifs mapped to the C-terminal domain (center) and their close-ups: ⁸⁸⁷EXNXDYIL⁸⁹⁴ (A), ⁹²⁵RXKXXLXXXFI⁹³⁵ (B), ⁹⁹⁶G—¹⁰⁰⁴PXXS¹⁰⁰⁷ (C), and ¹⁰¹⁶KKXXXXXK¹⁰²³ (D). Residues constituting the motifs and appropriate contact partners are labeled. The α-helices are numbered as in Fig. 1B. The domain coloring scheme is the same as in Fig. 2. Contacts are shown with dotted orange lines. The figure was prepared in YASARA.