Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV.
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ABSTRACT:
SUBMITTER: Ivanov I
PROVIDER: S-EPMC1780068 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses (sNSVs) produces short capped primers for viral transcription by cleaving the host mRNAs. EN requires divalent metals as cofactors for nucleic acid substrates cleavage; however, the detailed mechanism of metal ion-dependent catalysis of ENs remains obscure. In this work, we reported the EN crystal structure of the Ebinur Lake virus (EBIV), an emerging mosquito-borne orthobunyavirus, and investigated its enzymatic properties and metal ion-based catalytic mechanism. In vitro biochemical data showed that EBIV EN is a specific RNA nuclease and prefers to cleave unstructured uridine-rich ssRNA. Structural comparison indicated that the overall structural architecture of EBIV EN is similar to that of other sNSV ENs, while the detailed active site configuration including the binding state of metal ions and the conformation of the LA/LB loop pair is different. Based on sequence conservation analysis, nine active site mutants were constructed, and seven crystal structures of them were determined. Mutations of active site residues associated with the two metal ions (Mn1 and Mn2) coordination abolished EN activity. Crystallographic analyses further revealed that none of these mutants bound two metal ions simultaneously in the active site. Importantly, we found that the perturbation of Mn1-coordination (metal site 1), resulted in the enhancement or elimination of Mn2-coordination (metal site 2). Taken together, our data provide structural evidence to support the two-metal-ion catalytic mechanism of EBIV EN and the correlation of metal binding at the two binding sites, which may be commonly shared by bunyaviruses or other sNSVs. IMPORTANCE The viral endonucleases (ENs) encoded by bunyaviruses and orthomyxoviruses play an essential role in initiating transcription by "snatching" capped primers from the host mRNAs. These ENs are metal-ion-dependent nucleases; however, the details of their catalytic mechanism remain elusive. Here, we reported high-resolution crystal structures of the wild-type and mutant ENs of a novel bunyavirus, the Ebinur Lake virus (EBIV), and revealed the structure and function relationship of EN. The EBIV EN exhibited differences in the details of active site structure compared to its homologues. Our data provided structural evidence to support a two-metal-ion catalytic mechanism of EBIV EN, and found the correlation of metal binding at both binding sites, which might reflect the dynamic structural properties that correlate to EN catalytic function. Taken together, our results revealed the structural characteristics of EBIV EN and made important implications for understanding the catalytic mechanism of cap-snatching ENs.
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Project description:Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single-turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of the metallonuclease mechanism. DNA association rate constants are approximately 100-fold higher in the presence of the catalytically nonsupportive Ca(II) versus the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM(2)S and EM(4)S, respectively) support cleavage. Interestingly, the cleavage rate for EM(2)S is approximately 100-fold slower than that displayed by EM(4)S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two-metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.
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Project description:Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg(2+) and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.
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