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

Yang Z, Horton JR, Maunus R, Wilson GG, Roberts RJ, Cheng X - Nucleic Acids Res. (2005)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine 1510 Clifton Road, Atlanta, GA 30322, USA.

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.

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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.
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fig3: 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.

Mentions: As revealed by a Dali structure comparison (33), a pairwise least squares superposition of HinP1I and MspI gives a root-mean-square deviation of 3.1 Å over 195 Cα pairs (Z-score of 15.2). This close structural similarity is not evident at the sequence level: guided by structural alignment, HinP1I shows only 14% amino acid sequence identity (34 out of 247 residues) to MspI (Figure 3). Among the 34 identical residues, 12 with hydrophobic side chains intercalate to form the hydrophobic core of the molecule; 3 are catalytic (E18, D62 and K83); 2 are potentially involved in specific DNA contacts (Q236 and K238), and another 2 in DNA phosphate contacts (K60 and S86); 3 glycines (G7, G14 and G225) might be structurally important for short turns; 2 charged residues form an intra-molecular salt bridge (E154 and R157); and 2 polar residues are involved in intra-molecular hydrogen bonds (Q180 and N192). Seven additional conserved charged residues of unknown function (K19, D23, K29, D46, D109, R117 and R168) are scattered upon the surface of the molecule (Figure 2B).


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

Yang Z, Horton JR, Maunus R, Wilson GG, Roberts RJ, Cheng X - Nucleic Acids Res. (2005)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074309&req=5

fig3: 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.
Mentions: As revealed by a Dali structure comparison (33), a pairwise least squares superposition of HinP1I and MspI gives a root-mean-square deviation of 3.1 Å over 195 Cα pairs (Z-score of 15.2). This close structural similarity is not evident at the sequence level: guided by structural alignment, HinP1I shows only 14% amino acid sequence identity (34 out of 247 residues) to MspI (Figure 3). Among the 34 identical residues, 12 with hydrophobic side chains intercalate to form the hydrophobic core of the molecule; 3 are catalytic (E18, D62 and K83); 2 are potentially involved in specific DNA contacts (Q236 and K238), and another 2 in DNA phosphate contacts (K60 and S86); 3 glycines (G7, G14 and G225) might be structurally important for short turns; 2 charged residues form an intra-molecular salt bridge (E154 and R157); and 2 polar residues are involved in intra-molecular hydrogen bonds (Q180 and N192). Seven additional conserved charged residues of unknown function (K19, D23, K29, D46, D109, R117 and R168) are scattered upon the surface of the molecule (Figure 2B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine 1510 Clifton Road, Atlanta, GA 30322, USA.

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.

Show MeSH