Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

Abstract

The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

Figure 1. AspBHI is a member of MspJI family.

(a) Schematic representation of AspBHI and members of MspJI family. The conserved region is shown in dark grey and insertions are shown in open boxes. (b) Sequence alignment of AspBHI and members of MspJI family. The AspBHI residue numbering is shown above the sequence alignment. The pairwise comparison of AspBHI and MspJI was shown previously. Amino acids highlighted are either invariant (white against black) among the five proteins or similar (white against grey) as defined by the following groupings: V, L, I, and M; F, Y, and W; K and R, E and D; Q and N; E and Q; D and N; S and T; and A, G, and P. Helices are labeled αA-αM; strands are labeled β1–β15 (strand β8 is subdivided into β8₁ and β8₂ owing to a discontinuity in this strand). (c) Distribution of averaged crystallographic thermal B factor per residue.

Figure 2. Structure of AspBHI.

(a) Four AspBHI monomers, A, B, C and D, form a tetramer. Molecules C and D have mobile C-terminal domains (indicated by a circle). (b) AspBHI tetramer, rotated ~90° from the view of panel (a). (c) For comparison, MspJI has an intact tetramer showing in a similar orientation of panel (a). (d) The disordered C-terminal domains of molecules C and D of AspBHI tetramer were located in the void space along the crystallographic 6-fold axis with a diameter of 100 Å. (e) Elution profile of AspBHI on Superdex 200™10/300 GL (GE Healthcare). The column buffer was 20 mM Tris-HCl (pH 7.5), 300 mM NaCl and 1 mM DTT, and 150 ng of AspBHI was loaded onto the column. The inset shows the standardization of the size exclusion column using a Gel Filtration Markers Kit for Protein Molecular Weights (SIGMA-ALDRICH, Cat. No. MWGF1000) at the time AspBHI was profiled using the same buffer. (f) Monomeric AspBHI contains two domains connected by a linker. (g) AspBHI has a discontinuity in strand β8 owing to the insertion of a 3₁₀ helix (right panel), whereas MspJI has a corresponding 20-residue-long curved strand β8 (left panel). Pairwise sequence alignment is shown above the panels. (h) The 3₁₀ helix of molecule A is involved in the dimer interface with the C-terminal helix αL of molecule B. The amino end of the 3₁₀ helix (Ala149 of molecule A) interacts with the carboxyl end of helix αL (Ser368 of molecule B). Arrows indicate helical dipoles.

Figure 3. A model of AspBHI in complex with DNA.

(a) Superimposition of the AspBHI N-terminal domain (in green) with the SRA domain of mouse UHRF1 (in yellow; PDB 3FDE). (b) The flipped 5mC nucleotide can be docked into the binding pocket of AspBHI. (c) Superimposition of the AspBHI C-terminal endonuclease domain (in green) and the HindIII–DNA complex (conserved secondary elements in yellow and additional in grey) (PDB 2E52). (d) The scissile phosphate group (shown as an orange ball) is near the proposed catalytic residues (Glu303 and Lys305 in AspBHI). The side chain of conserved Asp282 in AspBHI, pointing away from the active site, might undergo conformational change upon DNA binding. (e) A model of the AspBHI N-terminal domain docked with a DNA (taken from PDB 3FDE) containing a flipped 5mC (which is faded in the background). The opposite guanine is labeled. The Loop-B3 occupies the DNA minor groove 5′ to the 5mC, while the Loop-2B occupies the minor groove 3′ to the 5mC.

Figure 4. AspBHI variants and activity assays on modified plasmid and phage DNA substrates.

(a) SDS-PAGE analysis of partially purified His-tagged AspBHI WT and its variants after nickel-chelated affinity chromatography. Arrow indicates the AspBHI protein band. (b) Endonuclease activity assay on phage XP12 DNA containing 5mC. Three concentration of WT AspBHI (~0.57 pmoles, with 2-fold serial dilution) were used in the digestion. Mutant enzyme concentrations were estimated at 0.29 to 0.57 pmoles. The smearing may result from partial digestions of the phage DNA. We note that S41C protein tends to precipitate in conditions with <0.2 M NaCl. (c) Endonuclease activity assay on Dcm⁺ and M.HpaII modified pUC19 DNA.

Figure 5. S41A and S41C activity assays on methylated oligonucleotide substrates.

(a) Schematic diagram of the fully methylated oligonucleotide substrates (M = 5mC) used for analyzing possible cleavage products (P1–P5 shown in panel b). (b) Duplex oligonucleotides (20 ng) were incubated at 37°C for 2 hours with 0.5 μg (0.29 pmoles) of WT, S41A, or S41C. Products were resolved on a 20% TBE native PAGE gel and visualized with Sybr Gold staining. Inserted is a 10–20% gradient SDS-PAGE showing the proteins used for crystallization (Se-Met) and for activity (WT, S41A and S41C). NEB protein ladder was used as molecular weight markers.