Structural basis for the recognition of sulfur in phosphorothioated DNA

Abstract

There have been very few reports on protein domains that specifically recognize sulfur. Here we present the crystal structure of the sulfur-binding domain (SBD) from the DNA phosphorothioation (PT)-dependent restriction endonuclease ScoMcrA. SBD contains a hydrophobic surface cavity that is formed by the aromatic ring of Y164, the pyrolidine ring of P165, and the non-polar side chains of four other residues that serve as lid, base, and wall of the cavity. The SBD and PT-DNA undergo conformational changes upon binding. The S¹⁸⁷RGRR¹⁹¹ loop inserts into the DNA major groove to make contacts with the bases of the G_PSGCC core sequence. Mutating key residues of SBD impairs PT-DNA association. More than 1000 sequenced microbial species from fourteen phyla contain SBD homologs. We show that three of these homologs bind PT-DNA in vitro and restrict PT-DNA gene transfer in vivo. These results show that SBD-like PT-DNA readers exist widely in prokaryotes.

Fig. 1

ScoMcrA employs its SBD domain to recognize PT-DNA. a Streptomyces coelicolor ScoMcrA specifically associated with PT-DNA and hemi‒PT-DNA containing the G_PSGCC core sequence, with the sulfur atom in the R_p, but not in the S_p, stereo-specific configuration. Lane 1 serves as a control with no protein added. b Domain organization of ScoMcrA. c Structure of the full-length ScoMcrA dimer. The four domains of ScoMcrA, head, SBD, SRA, and HNH domains, are labeled. d Structure of a ScoMcrA protomer. e Structure of ScoMcrA‒SBD-SRA in complex with a 10 base pair PT-DNA. The DNA sequence is shown at the top, with the bases recognized by ScoMcrA highlighted in magenta. f Structure of ScoMcrA‒SBD in complex with an eight base pair PT-DNA. Sulfur-recognizing residues are shown in stick as well in surface representations. The “S¹⁸⁷RGRR¹⁹¹ loop” recognizing the bases of PT-DNA is colored in orange

Fig. 2

Residues interacting with the sulfur atom. a Close-up view of the sulfur-binding cavity on ScoMcrA-SBD. The sulfur atom of PT-DNA is recognized by the pyrolidine ring of P165, the β-methylene groups of Y164 and H116, the β-methyl group of A168, and the γ-methylene group of R117 by hydrophobic interactions, as well as by the guanidinium group of R117 through electrostatic interaction. Electrostatic interactions are indicated as magenta dashed lines. b The view in a was rotated 90° towards left. c Schematic summary of the interactions between ScoMcrA-SBD and PT-DNA. d Mutations of key sulfur-recognizing or base-contacting residues disrupted or decreased the association between ScoMcrA-SBD and PT-DNA as analyzed by the EMSA assay. The original EMSA gels are shown in Supplementary Fig. 9

Fig. 3

ScoMcrA-SBD undergoes conformational change upon recognition of PT-DNA. a In the PT-DNA-unbound state of ScoMcrA-SBD, the phenyl ring of the side-chain of ScoMcrA-Y164 covers the sulfur-binding cavity. b In the PT-DNA-bound state, the hydroxyphenyl group of Y164 is flipped open to allow the sulfur atom of PT-DNA to access the sulfur-binding cavity. c Comparing the PT-DNA-bound and PT-DNA-unbound states of ScoMcrA-SBD, the hydroxyphenyl group of Y164 is rotated 98° upon association of PT-DNA. d Association of PT-DNA also triggers a substantial conformational change of the “S¹⁸⁷RGRR¹⁹¹ loop” of ScoMcrA-SBD

Fig. 4

The sulfur atom rotates ~80° outward upon association with ScoMcrA-SBD. a Comparison between the SBD-bound and SBD-unbound strands of the same PT-DNA molecule in the structure of ScoMcrA-SBD in complex with PT-DNA. An 80° outward rotation of the sulfur atom can be observed, which makes it more suitable for accommodation by the sulfur-binding cavity on ScoMcrA-SBD. b Comparison between the free and SBD-bound PT-DNA also shows that the sulfur atom rotates 83° outward upon its recognition by ScoMcrA-SBD

Fig. 5

Phylogenetic tree for prokaryotic ScoMcrA-SBD homologs. The two numbers behind each phylum/class/order represent the number of species possessing ScoMcrA-SBD homologs and that whose genomes have been sequenced. The occurrence frequency of ScoMcrA-SBD homologs, which was calculated by dividing the number of species possessing ScoMcrA-SBD homologs by the number of sequenced species, for different phyla/classes/orders in the bacteria kingdom was indicated. Details please see Supplementary Table 2

Fig. 6

SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R_p, but not in the S_p, configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G_PSAAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica, which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT⁻) and that harboring the dnd gene cluster (PT⁺) into E. coli DH10B expressing various scomcrA homologs are shown