Structural basis underlying complex assembly and conformational transition of the type I R-M system

Abstract

Type I restriction-modification (R-M) systems are multisubunit enzymes with separate DNA-recognition (S), methylation (M), and restriction (R) subunits. Despite extensive studies spanning five decades, the detailed molecular mechanisms underlying subunit assembly and conformational transition are still unclear due to the lack of high-resolution structural information. Here, we report the atomic structure of a type I MTase complex (2M+1S) bound to DNA and cofactor S-adenosyl methionine in the "open" form. The intermolecular interactions between M and S subunits are mediated by a four-helix bundle motif, which also determines the specificity of the interaction. Structural comparison between open and previously reported low-resolution "closed" structures identifies the huge conformational changes within the MTase complex. Furthermore, biochemical results show that R subunits prefer to load onto the closed form MTase. Based on our results, we proposed an updated model for the complex assembly. The work reported here provides guidelines for future applications in molecular biology.

Keywords: EcoKI; MTase; crystal structure; type I R-M system.

Fig. 1.

Overall structure of the Tte-MTase complex bound to DNA and SAM. (A) Schematics of the domain architectures of the S and M subunits of the type I R-M system from T. tengcongensis. The dashed lines indicate the disordered regions in the crystal structure. (B) Ribbon diagram of the structure of two Tte-MTase complexes in complex with one DNA and two SAM molecules in the asymmetric unit shown in two different views. Same color code as in A. The two MTase complexes are related by a noncrystallographic twofold symmetry. The individual domains of S and M subunits have been labeled in the Upper and Lower panels, respectively. The SAM molecules are shown in space-filling presentation and colored in green.

Fig. S1.

(A) Two views of the structural superimposition of the two MTases complexes (one in different colors; the other one in silver) in the asymmetric unit. (B and C) The 2Fo − Fc electron density maps around DNA (B) and SAM (C) are shown as the blue meshes (contoured at 1.2σ).

Fig. 2.

Four-helix bundle interface between S and M subunits. (A) Ribbon diagram of the four-helix bundle structure consisting of the CR regions (light blue) from the S subunit and two C-helices (yellow and violet) from the M subunits. The key residues are shown in stick representation. (B–D) Detailed interactions within the four-helix bundle interface as highlighted by the dashed boxes in A. The hydrophobic interactions are shown in space-filling presentation (B and D). The hydrogen bonds are shown as thick black dashed lines (C). Same color code as in A. (E–G) SDS/PAGE results of the coexpression of the wild-type S subunit (no tag) and different M subunits (His tagged) as indicated for both Tte (E and G) and EcoKI (F) systems. The SDS/PAGE gels were stained with Coomassie blue.

Fig. S2.

The secondary structure prediction for the C-terminal regions of the M subunits from different type I R-M systems. The linker and C-Helix motifs are colored in red and pink, respectively. The Jpred4 server (www.compbio.dundee.ac.uk/jpred/) was used for the secondary structure prediction.

Fig. S3.

The intermolecular interactions between the S and M subunits in addition to the four-helix bundle interface. Same color code as in Fig. 1. The hydrogen bonds are shown by green dashed lines.

Fig. 3.

Intermolecular interactions between DNA and TRD. (A) Ribbon diagram of the DNA molecule bound to the TRD1 domains from the two S subunits in the asymmetric unit. The TRD1 domains are colored in blue and black, respectively. The two DNA strands are colored in black and salmon. (B) Schematic of the detailed interactions between TRD1s and DNA. Same color code as in A. The hydrogen bonds between protein and the sugar-phosphate backbone or the bases are shown in dashed and solid lines, respectively. (C–E) Interactions between one TRD1 domain and the bound DNA in three different regions. The hydrogen bonds are shown by green dashed lines.

Fig. S4.

(A) Structure superimposition of the Tte-TRD1+DNA with the TRD domains from other R-M systems: LlaGI-TRD+DNA (Left), LlaBIII-TRD+DNA (Middle), and MmeI-TRD+DNA (Right). The Tte-TRD1 and the bound DNA are in light blue and red, respectively. The other TRDs and bound DNAs are in cyan and black, respectively. (B) The TRD2 domains (light blue) in the asymmetric unit bind to neighboring symmetric DNA molecules (red) by using the similar DNA-binding surface to TRD1.

Fig. 4.

Conformational transition from open to closed. (A) Surface views of the open (Left) and closed (Right) structures of the type I MTases. The color code for proteins is the same as in Fig. 1. The DNA is shown in ribbon and red. Given the low-quality electron density, the M2 subunit of the open complex was modeled in a similar conformation to M1 by superimposing the C-helices of the two M subunits. The dashed arrow line indicates the dynamic nature of this subunit in the structure. (B) Two views of the conformational changes in the S subunit between open (cyan) and closed (blue). The TRD1 domains (gray) are superimposed as the reference point. (C) The conformational changes in the M subunit between open (yellow) and closed (brown). The DNA (red) is shown in surface presentation. The TRD1 domains are superimposed as the reference point.

Fig. S5.

(A) The negative-stain EM closed model of the EcoKI-MTase. Same color code for the proteins as in Fig. 1. The DNA is shown in black ribbon. The red circles indicate the C-helix regions in the wrong conformation. (B) The updated closed model of the Tte-MTase. The S and M subunits of the EcoKI enzyme in A were replaced by their Tte counterparts by superimposition. The red circle indicates the revised four-helix bundle region based on the crystal structure.

Fig. S6.

(A–C) Size-exclusion chromatography curves (Upper) of different incubation samples and the related SDS/PAGE results (Lower) for the fractions from 10 mL to 16.5 mL (0.5 mL for each fraction).

Fig. S7.

(A–F) Size-exclusion chromatography curves of different incubation samples of the EcoKI system. (G) GST pull-down results showing that the R subunit bound to the Mtase complex in its closed state (with DNA) but not in the open state (no DNA).

Fig. 5.

The working model. The proposed model for the complex assembly and conformational changes of the type I R-M systems. The REase structure is modeled based on the crystal structure of the EcoR124I-R subunit (18) and the low-resolution EM model of EcoKI REase (11).