Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI

Abstract

HinP1I, a type II restriction endonuclease, recognizes and cleaves a palindromic tetranucleotide sequence (G/CGC) in double-stranded DNA, producing 2 nt 5' overhanging ends. Here, we report the structure of HinP1I crystallized as one protein monomer in the crystallographic asymmetric unit. HinP1I displays an elongated shape, with a conserved catalytic core domain containing an active-site motif of SDX18QXK and a putative DNA-binding domain. Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C/CGG) and binds to that sequence crystallographically as a monomer. Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements. Examining the protein-protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer. A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.

Figure 1

Dimeric form of endonucleases: (A) EcoRI–DNA complex (PDB 1CKQ), (B) EcoRV–DNA complex (PDB 1RVA), (C) BglI–DNA complex (PDB 1DMU). (D) Monomeric form of endonuclease: MspI–DNA complex (PDB 1SA3).

Figure 2

Structure of HinP1I (A) Ribbon representation. Helices are labeled as letters A–H and strands labeled as numbers 1–11 from N- to C-termini. Putative catalytic residues are shown in magenta color. The C-terminal β-hairpin, colored in red, contains the invariant residues (presumably involved in DNA base specific interactions) between HinP1I and MspI. (B) Molecular surface representation, shown in the same orientation of (A). The surface is colored blue for positive, red for negative and white for neutral. The basic (blue) concave surface, shown on the right side of the molecule, represents a DNA-binding surface. Several conserved charged residues (labeled) are scattered throughout the surface. R168, shown on the left side of the molecule, represents a potential dimer interface (see Figure 5B). (C) Superimposition of HinP1I (green) and MspI (cyan). (D) A model of HinP1I monomer docked with DNA.

Figure 3

Structure-based sequence alignment of HinP1I and MspI. The residue number and secondary structural elements of HinP1I and MspI are shown, respectively, above and below the aligned sequences. The amino acids highlighted are invariant (white letter against black background) and conserved (black against cyan). The letters immediately above or below the sequences indicate the structural and suggested functional roles of the corresponding residues: ‘B’ indicates DNA base interaction, ‘C’ indicates catalysis, ‘D’ indicates dimer interface, ‘H’ indicates hydrophobic core, ‘I’ indicates intra-molecule interaction, ‘P’ indicates DNA phosphate interaction and ‘S’ indicates conserved surface residues (whose locations are shown in Figure 2B). The protein–DNA interactions involving main chain atoms are not indicated.

Figure 4

Structural similarity between HinP1I and MspI (A) Schematic diagram of MspI–DNA interactions, reproduced and modified from Xu et al. (5). Solid lines indicate direct hydrogen bonds, dotted lines indicate water-mediated hydrogen bonds and ‘mc’ indicates interaction involving main chain atom. (B) Residues potentially important for DNA base specific recognition: superimposition of residues of HinP1I (green) and MspI (cyan). (C) Residues potentially important for catalysis: superimposition of active site residues of HinP1I (green), MspI (cyan) and EcoRV (grey). Four residues belong to a common motif of E……PDX₁₅DXK (EcoRV), E……SDX₁₈QXK (HinP1I) and E……TDX₁₇NXK (MspI).

Figure 5

Potential link between the dimeric form of HinP1I and the tetrameric restriction enzymes (A) The HinP1I dimer interface mediated by a crystallographic 2-fold symmetry. (B) The enlarged dimer interface of HinP1I consists of residues from helices αG and αF. (C) A model of HinP1I dimer docked with two DNA molecules. (D) Structure of a tetramer of the NgoMIV restriction endonuclease in complex with two DNA molecules (PDB 1FIU). Two primary dimers (blue and green) are positioned back-to-back to each other.

Figure 6

HinP1I solution properties (A) Overlay of chromatographs from elution experiments on a Superdex 75 HR 10/30 sizing column. Blue: elution of three molecular weight standard proteins, cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa) and BSA (66.0 kDa). Red: elution of HinPI1 after loading 100 μl at ∼3.7 mg/ml concentration. Magenta: elution of HinPI1 after loading 100 μl at ∼75 mg/ml concentration. (B) HinPI1 in the presence of increasing amounts of glutaraldehyde. HinPI1 (∼1.9 mg/ml) was treated with glutaraldehyde and incubated at room temperature for 1 h (total volume 20 μl). The reaction was stopped by the addition of 10 μl of 1 M glycine. An aliquot of 15 μl of 3× loading buffer was added to these samples and subsequently 20 μl of this final solution was loaded onto a 13% SDS–PAGE gel (Coomassie stain).

Figure 7

A hypothetical model of a HinP1I dimer bound with a single copy of DNA. The two monomers of Hinp1I are shown in green and magenta. (A) The presentation of green monomer of Hinp1I is similar to that shown in Figure 2D. (B) A view looking into the DNA major groove.