Limits...
Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion.

Korada SK, Johns TD, Smith CE, Jones ND, McCabe KA, Bell CE - Nucleic Acids Res. (2013)

Bottom Line: Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation.The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases.The structures provide new insights into the mechanism of processive digestion that will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Avenue, Columbus, OH 43210, USA.

ABSTRACT
Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3'-5' direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3'-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an 'anchor' site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.

Show MeSH

Related in: MedlinePlus

Overlay of complexes A and B observed in the Cy5-dT13 structure. (A) Complexes A (cyan) and B (magenta) were superimposed using all protein Cα atoms (rmsd 1.3 Å). Notice that all 13 nucleotides of the ssDNA are bound within the ExoI groove in complex A, whereas in complex B, only the first 10 nucleotides are bound to the groove, such that the three 5′ nucleotides extend from the surface of the complex. The 3′-nucleotide in the active site and the three nucleotides in the anchor site of each complex overlap closely, whereas those in the bulge region differ significantly. (B) Close-up view of the interaction with the bulge region. A portion of the SH3-like domain of complex B rotates inward by ∼4 Å to maintain its contact with the shorter bulge region. The superposition is the same as in panel A, but zoomed in on the region that shows the largest difference in the conformation of the protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675492&req=5

gkt278-F3: Overlay of complexes A and B observed in the Cy5-dT13 structure. (A) Complexes A (cyan) and B (magenta) were superimposed using all protein Cα atoms (rmsd 1.3 Å). Notice that all 13 nucleotides of the ssDNA are bound within the ExoI groove in complex A, whereas in complex B, only the first 10 nucleotides are bound to the groove, such that the three 5′ nucleotides extend from the surface of the complex. The 3′-nucleotide in the active site and the three nucleotides in the anchor site of each complex overlap closely, whereas those in the bulge region differ significantly. (B) Close-up view of the interaction with the bulge region. A portion of the SH3-like domain of complex B rotates inward by ∼4 Å to maintain its contact with the shorter bulge region. The superposition is the same as in panel A, but zoomed in on the region that shows the largest difference in the conformation of the protein.

Mentions: Based on a structural superposition (Figure 3 and Supplementary Table S1), complexes A and B differ mainly in the conformation of the DNA, as opposed to the protein. The protein portions of the two complexes superimpose to an rmsd of 1.3 Å for all Cα atoms. The structural differences do not involve large-scale domain movements, but instead are primarily confined to a segment of the SH3-like domain (residues 268–297) that moves inward by ∼4 Å in complex B to maintain its contact with the shorter bulge region (Figure 3B). There are also no large-scale domain movements that occur in ExoI on DNA binding. Complexes A and B align to previously reported structures of unbound ExoI to rmsd values ranging from 0.9 to 1.5 Å, and 1.4 to 1.8 Å, respectively, for all Cα atoms (Supplementary Table S1). Again, the most significant differences between DNA-bound and unbound ExoI structures occur in the segment of the SH3-like domain that contacts the bulge region of the DNA. In particular, residues 277–295 of this segment adopt a particularly closed conformation in the original ExoI structure (1FXX; 12), which would clash with the bulge region of the ssDNA in both complexes. However, in other crystal structures of uncomplexed ExoI, this segment of the SH3-like domain is disordered (13,21), suggesting that it is in general a highly flexible region of the structure, as opposed to one that adopts distinct open and closed states in the presence and absence of DNA, respectively.Figure 3.


Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion.

Korada SK, Johns TD, Smith CE, Jones ND, McCabe KA, Bell CE - Nucleic Acids Res. (2013)

Overlay of complexes A and B observed in the Cy5-dT13 structure. (A) Complexes A (cyan) and B (magenta) were superimposed using all protein Cα atoms (rmsd 1.3 Å). Notice that all 13 nucleotides of the ssDNA are bound within the ExoI groove in complex A, whereas in complex B, only the first 10 nucleotides are bound to the groove, such that the three 5′ nucleotides extend from the surface of the complex. The 3′-nucleotide in the active site and the three nucleotides in the anchor site of each complex overlap closely, whereas those in the bulge region differ significantly. (B) Close-up view of the interaction with the bulge region. A portion of the SH3-like domain of complex B rotates inward by ∼4 Å to maintain its contact with the shorter bulge region. The superposition is the same as in panel A, but zoomed in on the region that shows the largest difference in the conformation of the protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675492&req=5

gkt278-F3: Overlay of complexes A and B observed in the Cy5-dT13 structure. (A) Complexes A (cyan) and B (magenta) were superimposed using all protein Cα atoms (rmsd 1.3 Å). Notice that all 13 nucleotides of the ssDNA are bound within the ExoI groove in complex A, whereas in complex B, only the first 10 nucleotides are bound to the groove, such that the three 5′ nucleotides extend from the surface of the complex. The 3′-nucleotide in the active site and the three nucleotides in the anchor site of each complex overlap closely, whereas those in the bulge region differ significantly. (B) Close-up view of the interaction with the bulge region. A portion of the SH3-like domain of complex B rotates inward by ∼4 Å to maintain its contact with the shorter bulge region. The superposition is the same as in panel A, but zoomed in on the region that shows the largest difference in the conformation of the protein.
Mentions: Based on a structural superposition (Figure 3 and Supplementary Table S1), complexes A and B differ mainly in the conformation of the DNA, as opposed to the protein. The protein portions of the two complexes superimpose to an rmsd of 1.3 Å for all Cα atoms. The structural differences do not involve large-scale domain movements, but instead are primarily confined to a segment of the SH3-like domain (residues 268–297) that moves inward by ∼4 Å in complex B to maintain its contact with the shorter bulge region (Figure 3B). There are also no large-scale domain movements that occur in ExoI on DNA binding. Complexes A and B align to previously reported structures of unbound ExoI to rmsd values ranging from 0.9 to 1.5 Å, and 1.4 to 1.8 Å, respectively, for all Cα atoms (Supplementary Table S1). Again, the most significant differences between DNA-bound and unbound ExoI structures occur in the segment of the SH3-like domain that contacts the bulge region of the DNA. In particular, residues 277–295 of this segment adopt a particularly closed conformation in the original ExoI structure (1FXX; 12), which would clash with the bulge region of the ssDNA in both complexes. However, in other crystal structures of uncomplexed ExoI, this segment of the SH3-like domain is disordered (13,21), suggesting that it is in general a highly flexible region of the structure, as opposed to one that adopts distinct open and closed states in the presence and absence of DNA, respectively.Figure 3.

Bottom Line: Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation.The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases.The structures provide new insights into the mechanism of processive digestion that will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Avenue, Columbus, OH 43210, USA.

ABSTRACT
Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3'-5' direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3'-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an 'anchor' site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.

Show MeSH
Related in: MedlinePlus