Fig 1.
Crystal Structure of MpAgo bound to a RNA guide and DNA target heteroduplex.
(A) Domain order of MpAgo with residue number demarcation. A cartoon representation of the MpAgo crystal structure with labeled N-terminal (green), Linker L1 (grey), PAZ (pink), Linker L2 (yellow), MID (purple), and PIWI (blue) domains bound to a 5′-hydroxylated 21 nt guide RNA (orange) and 5′-phosphorylated 21 nt target DNA (red). The sequence of the guide and target are aligned with black dots representing Watson-Crick base-pairing and unmodeled nucleotides are colored grey. (B) Surface representation of MpAgo with the guide RNA (orange) and target DNA (red) heteroduplex bound in-between the N- and C- lobes.
Table 1.
Data collection and refinement statistics of MpAgo ternary complex
Fig 2.
Conformational changes between MpAgo binary and ternary complexes.
(A) A transparent cartoon representation of MpAgo bound to guide RNA only (PDB ID: 5I4A) with vector arrows, generated using PyMol, indicating conformational changes of MpAgo upon target binding. Black arrows represent the vector direction of the Linker L2, PAZ domain, and N domain away from the C-lobe. (B) The guide RNA (blue) from the MpAgo binary complex is overlaid with the guide RNA (orange) from the MpAgo ternary complex after alignment of the PIWI domains from the two structures. The black arrows show direction of conformational changes of the guide RNA upon target DNA (transparent red) binding.
Fig 3.
The N-terminal domain orientation of MpAgo stabilizes the RNA-DNA heteroduplex in a linear conformation.
(A) MpAgo (colored by domain) was aligned to RsAgo (PDB ID: 5AWH, grey) relative to their PIWI domains. The MpAgo RNA guide (orange) and DNA target (red) heteroduplex remains in a linear conformation between the two MpAgo lobes. After two helical turns, the RNA guide (blue) and DNA target (blue) of RsAgo angles behind the PIWI domain and away from the PAZ domain. (B) The Argonaute structures from Fig 1A were superimposed and cropped to focus on the N-terminal domains and helices. The unique orientation of the MpAgo N-terminal domain (green) close to the PAZ domain corresponds with the linear conformation of the heteroduplex (red), while the angled N-terminal domain of RsAgo (grey) appears to bend the heteroduplex behind the PIWI domain. α-helix 1 of each domain is labeled to highlight the dramatic change in orientation relative to α-helix 3. (C) A cartoon representation of the linear heteroduplex of MpAgo (red) and the bent heteroduplex of RsAgo (blue), which occurs after the second helical turn.
Fig 4.
Symmetric mismatch tolerance across hybrid helix.
(A) Dinucleotide mismatches were introduced across the entire guide RNA starting at the 5′ end. MpAgo cleavage kinetics of a ssDNA target was measured and the 30 min time point was plotted against mismatch position. Each reaction reached completion after 30 min. Error bars represent SD of three independent experiments. (B) Cartoon representation of the MpAgo RNA guide and DNA target heteroduplex. Heatmap coloring of the helix displays where dinucleotide mismatches inhibit DNA cleavage efficiency (white) or are tolerated (red).