5g5t
From Proteopedia
Structure of the Argonaute protein from Methanocaldcoccus janaschii in complex with guide DNA
Structural highlights
FunctionAGO_METJA A DNA-guided ssDNA endonuclease that may play a role in defense against invading genetic elements. Uses short ssDNA sequences as guides (gDNA) to bind complementary target strands, resulting in slicing of the target DNA (tDNA) (PubMed:24442234, PubMed:27741323, PubMed:28319081, PubMed:28319084). Endonucleolytically cleaves tDNA (the gDNA indicates where to cleave); two major and two minor products are seen which correspond to cleavage sites between nucleotides 9/10, 10/11, 13/14, and 14/15 downstream of the target residue base-paired with the 5'-end of the gDNA (PubMed:24442234, PubMed:27741323, PubMed:28319081, PubMed:28319084). Efficient guide-dependent tDNA cleavage requires a minimal length of 15 bp and is maximal at 19 bp (PubMed:28319081). Prefers gDNA with 5'-phosphorylated purines and 3'-pyrimidines; changing these bases alters the cleavage activity and patterns (PubMed:28319084). Also has guide-independent activity on tDNA called 'chopping' (PubMed:28319081). Probably a first round of guide-independent activity on an invading plasmid or virus would generate guide DNAs for subsequent, more efficient, guide-dependent degradation of invading nucleic acids (PubMed:28319081). Has no activity on substrate with a mismatch at positions 10 and 11, on ssDNA or RNA, nor on DNA:RNA hybrids (PubMed:24442234). Digests longer (750 bp) dsDNA as well as circular plasmid and naked genomic DNA, but not chromatin, in a guide DNA-independent manner (PubMed:28319081). Addition of endogenous histone A3 protects DNA from cleavage, while cleavage is insensitive to methylation (PubMed:28319081). When plasmid encoding active or mutated protein (Ala-541) is transformed into Sulfolobus acidocaldarius about 25-fold fewer transformants are found with active protein; reduced levels of plasmid are found in wild-type transformed cells. While S.acidocaldarius grows at a similar temperature to M.jannaschii (70 to 80 degrees Celsius) it has very different histone-like proteins, which presumably do not protect against MjAgo (PubMed:28319081). Binds ssDNA, dsDNA and DNA-RNA hybrids; binding is most efficient with dsDNA (PubMed:24442234).[1] [2] [3] [4] [5] Publication Abstract from PubMedArgonaute (Ago) proteins in eukaryotes are known as key players in post-transcriptional gene silencing1, while recent studies on prokaryotic Agos hint at their role in the protection against invading DNA2,3. Here, we present crystal structures of the apo enzyme and a binary Ago-guide complex of the archaeal Methanocaldococcus jannaschii (Mj) Ago. Binding of a guide DNA leads to large structural rearrangements. This includes the structural transformation of a hinge region containing a switch helix, which has been shown for human Ago2 to be critical for the dynamic target search process4-6. To identify key residues crucial for MjAgo function, we analysed the effect of several MjAgo mutants. We observe that the nature of the 3' and 5' nucleotides in particular, as well as the switch helix, appear to impact MjAgo cleavage activity. In summary, we provide insights into the molecular mechanisms that drive DNA-guided DNA silencing by an archaeal Ago. Structural and mechanistic insights into an archaeal DNA-guided Argonaute protein.,Willkomm S, Oellig CA, Zander A, Restle T, Keegan R, Grohmann D, Schneider S Nat Microbiol. 2017 Mar 20;2:17035. doi: 10.1038/nmicrobiol.2017.35. PMID:28319084[6] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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