Structural insights to the metal specificity of an archaeal member of the LigD 3′-phosphoesterase DNA repair enzyme family

Abstract

LigD 3′-phosphoesterase (PE) enzymes perform end-healing reactions at DNA breaks. Here we characterize the 3′-ribonucleoside-resecting activity of Candidatus Korarchaeum PE. CkoPE prefers a single-stranded substrate versus a primer–template. Activity is abolished by vanadate (10 mM), but is less sensitive to phosphate (IC50 50 mM) or chloride (IC50 150 mM). The metal requirement is satisfied by manganese, cobalt, copper or cadmium, but not magnesium, calcium, nickel or zinc. Insights to CkoPE metal specificity were gained by solving new 1.5 Å crystal structures of CkoPE in complexes with Co2+ and Zn2+. His9, His15 and Asp17 coordinate cobalt in an octahedral complex that includes a phosphate anion, which is in turn coordinated by Arg19 and His51. The cobalt and phosphate positions and the atomic contacts in the active site are virtually identical to those in the CkoPE·Mn2+ structure. By contrast, Zn2+ binds in the active site in a tetrahedral complex, wherein the position, orientation and atomic contacts of the phosphate are shifted and its interaction with His51 is lost. We conclude that: (i) PE selectively binds to ‘soft’ metals in either productive or non-productive modes and (ii) PE catalysis depends acutely on proper metal and scissile phosphate geometry.

RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a

“…Even though Mg 2+ can form an octahedral coordination sphere, Mn 2+ and Co 2+ may promote optimal orientation of the DNA in the active site to catalyze DNA cleavage in the absence of a guide RNA. This observation is supported by the crystal structures of DNA ligase D 3′-phosphoesterase (PE) enzyme, where Mn 2+ and Co 2+ promote a productive orientation of the metal, phosphate, and active site residues of the protein, but Zn 2+ does not ( Das et al, 2012 ). Many Mn 2+ -dependent DNA repair enzymes are active specifically in the presence of Mn 2+ , but not with Mg 2+ ( Das et al, 2012 ; Zhu and Shuman, 2005 ), because of reduced transition state stability in the presence of Mg 2+ .…”

“…This observation is supported by the crystal structures of DNA ligase D 3′-phosphoesterase (PE) enzyme, where Mn 2+ and Co 2+ promote a productive orientation of the metal, phosphate, and active site residues of the protein, but Zn 2+ does not ( Das et al, 2012 ). Many Mn 2+ -dependent DNA repair enzymes are active specifically in the presence of Mn 2+ , but not with Mg 2+ ( Das et al, 2012 ; Zhu and Shuman, 2005 ), because of reduced transition state stability in the presence of Mg 2+ . In the case of DNA polymerases, divalent metal ions such as Mn 2+ , Co 2+ , Ni 2+ , and Zn 2+ can substitute for Mg 2+ , with a subsequent reduction in fidelity and processivity of the enzyme ( Frank and Woodgate, 2007 ).…”

Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D

“…A crystal structure of Cko PE bound to Zn 2+ and phosphate has recently been determined ( 35 ). The authors found that the structure of Cko PE•Zn 2+ •phosphate complex is essentially identical to that of the previously determined Cko PE•Mn 2+ •sulfate complex.…”

“…These differences cannot be explained simply by an alteration of the co-ordination sphere of the metal, rather points to a significant conformational change involving a large part of the catalytic face. While it is possible that the conformational changes induced by Zn 2+ are different for the Pae PE and Cko PE domains [Zn 2+ acts as inhibitor for both proteins ( 12 , 35 )], it is more likely that the conformers that display these changes are unstable under crystal packing forces.…”

Prediction of models for ordered solvent in macromolecular structures by a classifier based upon resolution-independent projections of local feature data

“…Common sources include the chemical environment used to grow the macromolecular crystals and the solution components in which the macromolecules themselves are isolated. However, solvent species can appear in structures as a result of carry-over from upstream purification or contamination from common laboratory reagents (Niedzialkowska et al, 2016;Das et al, 2012). Thus, a list of chemical species that are explicitly present during crystal growth does not provide an exhaustive list of chemical species that should be considered when model building.…”