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Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI.

Sasnauskas G, Zagorskaitė E, Kauneckaitė K, Tamulaitiene G, Siksnys V - Nucleic Acids Res. (2015)

Bottom Line: Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5'-C(mC)DG-3' target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G).Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy.Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania gsasnaus@ibt.lt.

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DNA recognition by SRA domains. The structures of the DNA-bound UHRF1-SRA and MspJI, and the apo-structures of MspJI, LpnPI-N and AspBHI-N (PDB ID: 3FDE, 4R28, 4F0Q, 4RZL, 4OC8) were superimposed with MultiProt (29). Equivalent DNA recognition elements in all panels are shown in identical orientation. Left: recognition of the flipped-out base in the protein pocket; center: Loop-6C or ‘CpG recognition’/‘NKR finger’ loop (cyan); right: Loop-B3 or ‘base-flipping-promotion’ loop (blue), Loop-B2 (magenta), and Loop-78 (green). In all panels the flipped-out base and the orphan intra-helical guanine are colored red; other nucleotides comprising the specific recognition sequence of the corresponding protein are colored orange and are numbered from ‘−2’ (the second bp upstream of 5mC) to ‘+3’ (the third bp downstream of 5mC). (A) DNA recognition by MspJI. Loop-6C occupies a similar position both in the apo- and the DNA-bound structures and does not make base-specific contacts. Residues Q33, E65, and K173 from the ‘2B’, ‘B3’, and ‘78’ loops, respectively, are close to the DNA bases. (B and C) The models of DNA-bound LpnPI and AspBHI based on the co-crystal structure of UHRF1-SRA. Loop-C6 and Loop-B3 residues 41–43, 91–93, and 99 are different in LpnPI and AspBHI. LpnPI Loop-2B and Loop-78 residues 27–29 and 136–137 were mutated in the present study; AspBHI Loop-2B residues T25 and D32 are critical for the enzyme function (13); AspBHI Loop-78 residue R132 overlaps with the critical LpnPI residue R137. (D) DNA recognition by the SRA domain of the eukaryotic UHRF1 protein. The loops equivalent to Loop-78 and Loop-2B in MspJI-like restriction endonucleases are colored green and magenta, respectively.
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Figure 2: DNA recognition by SRA domains. The structures of the DNA-bound UHRF1-SRA and MspJI, and the apo-structures of MspJI, LpnPI-N and AspBHI-N (PDB ID: 3FDE, 4R28, 4F0Q, 4RZL, 4OC8) were superimposed with MultiProt (29). Equivalent DNA recognition elements in all panels are shown in identical orientation. Left: recognition of the flipped-out base in the protein pocket; center: Loop-6C or ‘CpG recognition’/‘NKR finger’ loop (cyan); right: Loop-B3 or ‘base-flipping-promotion’ loop (blue), Loop-B2 (magenta), and Loop-78 (green). In all panels the flipped-out base and the orphan intra-helical guanine are colored red; other nucleotides comprising the specific recognition sequence of the corresponding protein are colored orange and are numbered from ‘−2’ (the second bp upstream of 5mC) to ‘+3’ (the third bp downstream of 5mC). (A) DNA recognition by MspJI. Loop-6C occupies a similar position both in the apo- and the DNA-bound structures and does not make base-specific contacts. Residues Q33, E65, and K173 from the ‘2B’, ‘B3’, and ‘78’ loops, respectively, are close to the DNA bases. (B and C) The models of DNA-bound LpnPI and AspBHI based on the co-crystal structure of UHRF1-SRA. Loop-C6 and Loop-B3 residues 41–43, 91–93, and 99 are different in LpnPI and AspBHI. LpnPI Loop-2B and Loop-78 residues 27–29 and 136–137 were mutated in the present study; AspBHI Loop-2B residues T25 and D32 are critical for the enzyme function (13); AspBHI Loop-78 residue R132 overlaps with the critical LpnPI residue R137. (D) DNA recognition by the SRA domain of the eukaryotic UHRF1 protein. The loops equivalent to Loop-78 and Loop-2B in MspJI-like restriction endonucleases are colored green and magenta, respectively.

Mentions: The flipped-out cytosine binding pockets are similar in all SRA domains (Figure 2). The cytosine 5-methyl group in MspJI pocket is in van der Waals distance from D117, Y114 and W101 residues, and apparently makes a weak C–H…O hydrogen bond to the carbonyl oxygen of G116. These interactions may serve to distinguish modified cytosine from an unmodified base (18). Equivalent positions in LpnPI and AspBHI are occupied by D85, Y82, W69, and G84 residues. The side walls of the MspJI pocket are formed by the residues W101, Y114 and D117, while D103, S90 and F115 make hydrogen bonds to the Watson-Crick edge of the modified cytosine. Equivalent residues in LpnPI are W69, Y82, D85, K87, D71, N63 and Y83 (W69, Y82, D85, R87, D71, N63, Y83 in AspBHI). Mutation of the AspBHI pocket residues D71, Y82 and D85 to alanine abolished the DNA cleavage activity (13).


Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI.

Sasnauskas G, Zagorskaitė E, Kauneckaitė K, Tamulaitiene G, Siksnys V - Nucleic Acids Res. (2015)

DNA recognition by SRA domains. The structures of the DNA-bound UHRF1-SRA and MspJI, and the apo-structures of MspJI, LpnPI-N and AspBHI-N (PDB ID: 3FDE, 4R28, 4F0Q, 4RZL, 4OC8) were superimposed with MultiProt (29). Equivalent DNA recognition elements in all panels are shown in identical orientation. Left: recognition of the flipped-out base in the protein pocket; center: Loop-6C or ‘CpG recognition’/‘NKR finger’ loop (cyan); right: Loop-B3 or ‘base-flipping-promotion’ loop (blue), Loop-B2 (magenta), and Loop-78 (green). In all panels the flipped-out base and the orphan intra-helical guanine are colored red; other nucleotides comprising the specific recognition sequence of the corresponding protein are colored orange and are numbered from ‘−2’ (the second bp upstream of 5mC) to ‘+3’ (the third bp downstream of 5mC). (A) DNA recognition by MspJI. Loop-6C occupies a similar position both in the apo- and the DNA-bound structures and does not make base-specific contacts. Residues Q33, E65, and K173 from the ‘2B’, ‘B3’, and ‘78’ loops, respectively, are close to the DNA bases. (B and C) The models of DNA-bound LpnPI and AspBHI based on the co-crystal structure of UHRF1-SRA. Loop-C6 and Loop-B3 residues 41–43, 91–93, and 99 are different in LpnPI and AspBHI. LpnPI Loop-2B and Loop-78 residues 27–29 and 136–137 were mutated in the present study; AspBHI Loop-2B residues T25 and D32 are critical for the enzyme function (13); AspBHI Loop-78 residue R132 overlaps with the critical LpnPI residue R137. (D) DNA recognition by the SRA domain of the eukaryotic UHRF1 protein. The loops equivalent to Loop-78 and Loop-2B in MspJI-like restriction endonucleases are colored green and magenta, respectively.
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Figure 2: DNA recognition by SRA domains. The structures of the DNA-bound UHRF1-SRA and MspJI, and the apo-structures of MspJI, LpnPI-N and AspBHI-N (PDB ID: 3FDE, 4R28, 4F0Q, 4RZL, 4OC8) were superimposed with MultiProt (29). Equivalent DNA recognition elements in all panels are shown in identical orientation. Left: recognition of the flipped-out base in the protein pocket; center: Loop-6C or ‘CpG recognition’/‘NKR finger’ loop (cyan); right: Loop-B3 or ‘base-flipping-promotion’ loop (blue), Loop-B2 (magenta), and Loop-78 (green). In all panels the flipped-out base and the orphan intra-helical guanine are colored red; other nucleotides comprising the specific recognition sequence of the corresponding protein are colored orange and are numbered from ‘−2’ (the second bp upstream of 5mC) to ‘+3’ (the third bp downstream of 5mC). (A) DNA recognition by MspJI. Loop-6C occupies a similar position both in the apo- and the DNA-bound structures and does not make base-specific contacts. Residues Q33, E65, and K173 from the ‘2B’, ‘B3’, and ‘78’ loops, respectively, are close to the DNA bases. (B and C) The models of DNA-bound LpnPI and AspBHI based on the co-crystal structure of UHRF1-SRA. Loop-C6 and Loop-B3 residues 41–43, 91–93, and 99 are different in LpnPI and AspBHI. LpnPI Loop-2B and Loop-78 residues 27–29 and 136–137 were mutated in the present study; AspBHI Loop-2B residues T25 and D32 are critical for the enzyme function (13); AspBHI Loop-78 residue R132 overlaps with the critical LpnPI residue R137. (D) DNA recognition by the SRA domain of the eukaryotic UHRF1 protein. The loops equivalent to Loop-78 and Loop-2B in MspJI-like restriction endonucleases are colored green and magenta, respectively.
Mentions: The flipped-out cytosine binding pockets are similar in all SRA domains (Figure 2). The cytosine 5-methyl group in MspJI pocket is in van der Waals distance from D117, Y114 and W101 residues, and apparently makes a weak C–H…O hydrogen bond to the carbonyl oxygen of G116. These interactions may serve to distinguish modified cytosine from an unmodified base (18). Equivalent positions in LpnPI and AspBHI are occupied by D85, Y82, W69, and G84 residues. The side walls of the MspJI pocket are formed by the residues W101, Y114 and D117, while D103, S90 and F115 make hydrogen bonds to the Watson-Crick edge of the modified cytosine. Equivalent residues in LpnPI are W69, Y82, D85, K87, D71, N63 and Y83 (W69, Y82, D85, R87, D71, N63, Y83 in AspBHI). Mutation of the AspBHI pocket residues D71, Y82 and D85 to alanine abolished the DNA cleavage activity (13).

Bottom Line: Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5'-C(mC)DG-3' target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G).Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy.Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania gsasnaus@ibt.lt.

Show MeSH