Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification

DNA phosphorothioate (PT) modification, originally developed as an artificial tool to stabilize oligodeoxynucleotides against nuclease degradation¹, was recently found to be incorporated with sulfur into DNA backbone as a novel physiological variation by the five-gene dnd cluster (dndA-dndE) products in a sequence- and stereo-specific manner². This PT modification causes the DNA degradation (Dnd) phenotype and is widespread and quantized in bacterial genomes, working as a part of a restriction modification system^3,4,5. This modification can be specifically cleaved in vitro by type IV restriction endonuclease⁶. DndA works as a cysteine desulfurase and assembles DndC as a 4Fe-4S cluster protein⁷. DndC possesses ATP pyrophosphatase activity^8,9 and is predicted to have 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase activity, whereas DndB has homology to a group of transcriptional regulators^10,11. DndD, known as SpfD in Pseudomonas fluorescens Pf0-1, has ATPase activity possibly related to DNA structure alteration or nicking during PT incorporation^5,12. Sequence identity (46%) and similarity (61%) to phosphoribosylaminoimidazole carboxylase (NCAIR synthetase) from Anabaena variabilis¹¹ suggest that DndE could be a NCAIR synthase analogue¹³. However, DndE may also act as a sulfotransferase due to a specific PAPS binding sequence AAVGK-TLLIHLHR contained in the C-terminus of DndE from Streptomyces lividans (DndE_strep)¹⁰. Therefore, the exact function of DndE remains unknown.

The DndE_B7A-N110 structure reveals a tetramer conformer (Figure 1B), consistent with the results from DLS assay. The tetramer adopts a quadrate fold, resembling a four-leaf clover of 62 Å in both length and width and 32 Å in height. It contains two kinds of dimers (A-E and A-M) with different stability resulted from different numbers of H-bonds in the dimer interface (Figure 1C and 1D). Two clefts (∼60° and ∼90°) are formed by the dimers (Figure 1E and 1F). Electrostatic surface analysis indicates that the cleft-containing side is more hydrophobic than its opposite side (Figure 1G and 1H). The K20 side-chain in each monomer extends into the tetramer center, forming hydrogen bonds with G24 and/or G21 in the next monomer, producing a large positively charged hole (Figure 1B-1D and 1G). The four monomer structures are almost identical and can be superimposed with an root-mean-square deviation of 0.33 Å over 59 backbone Cα atoms in the secondary structural region. Each monomer comprises five helices: H1 (aa 10-22), H2 (aa 27-39), H3 (aa 62-66), H4 (aa 70-77) and H5 (aa 86-102) (Figure 1A). Together with the H2 helix, a long flexible loop (aa 42-60) between the H2 and H3 helices encloses the H5 helix, where the residues R49, D50, S51 and K52 are not observable in some monomers.

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