Structure, Volume 24

Supplemental Information

Structure of the EndoMS-DNA Complex

as Mismatch Restriction Endonuclease

Setsu Nakae, Atsushi Hijikata, Toshiyuki Tsuji, Kouki Yonezawa, Ken-ichi Kouyama, KoutaMayanagi, Sonoko Ishino, Yoshizumi Ishino, and Tsuyoshi Shirai

SUPPLEMENTARY INFORMATION

Structure of the EndoMS-DNA complex as mismatch-restriction endonuclease

Setsu Nakae, Astuhi Hijikata, Toshiyuki Tsuji, Koki Yonezawa, Ken-ichi Kouyama, Kouta

Mayanagi, Sonoko Ishino, Yoshizumi Ishino, Tsuyoshi Shirai

Contents

Detailed Materials and Methods

Supplementary Figure S1. Related to Figure 2. Alignment of putative EndoMS orthologs

Supplementary Figure S2. Related to Figure 2. Molecular phylogeny and genome structure of putative EndoMS

orthologs

Supplementary Figure S3. Related to Figure 2. Structures of EndoMS and type II restriction enzymes

Supplementary Figure S4. Related to Figure 3. Sequence and affinity of DNAs for EndoMS substrates

Supplementary Figure S5. Related to Figure 3. DNA bound-structures of EndoMS

Supplementary Figure S6. Related to Figure 4. Electron microscopic images and class averages of the

TkoEndoMS-TkoPCNA- dsDNA complex

Supplementary Table S1. Related to Figure 2. Structures similar to N- or C-terminal domains of EndoMS in the

PDB

Supplementary Table S2. Related to Figure 3. DNA base-pair and base-step parameters of EndoMS-bound

and canonical B-form DNAs

Supplementary References

Detailed Materials and Methods

Database survey. Homologs of TkoEndoMS were screened using amino acid sequence (UniProt) and structure (PDB)

databases 1,2. The amino acid sequence of TkoEndoMS (UniProt entry Q5JER9) was used for the query, and the

UniProtKB database was searched with BLAST 3. A total of 1,336 sequences were found to have E-values lower than

0.01 and were aligned with ClustalW; their molecular phylogenies were constructed with the neighbor-joining method

4,5. Multiple sequence alignment and the phylogenetic tree of the 42 EndoMS/NucS homologs, which were selected

according to clustering on the tree and overall conservation between TkoEndoMS, are shown in Figures S1 and S2,

respectively. Putative EndoMS/NucS orthologs were detected in Archaea (Euryarchaeota, Crenarchaeota, and

Thaumarchaeota) and a group of Eubacteria (Actinobacteria and Deinococcus-Thermus).

The structural similarities between EndoMS/NucS and other proteins were examined in the PDB. Subunit A

of PabNucS (PDB: 2VLD) was divided into N-terminal (residues Lys3 to Glu120) and C-terminal (residues Ser126 to

Pro233) domains, and each domain was separately used for a query in a structural search with the fast structure

superposition application in the SIRD database system (http://sird.nagahama-i-bio.ac.jp/sird/). Subunits/domains that

showed less than 4.0 Å root mean square deviation (RMSD) for more than 50 residues were retrieved (Table S1).

Redundant hits on similar structures (more than 20% sequence identity) were discarded.

The results revealed that a few proteins showed structures similar to that of the N-terminal domain.

Although the proteins appeared to bind RNA, none of the structures of these proteins had been determined in complex

with RNA; thus, these proteins did not provide useful information for modeling of EndoMS-dsDNA interactions. In

contrast, many DNA-binding proteins have been detected for the C-terminal domain. This was expected because the

C-terminal domain of EndoMS belongs to a fold family, which contains a large variety of restriction, recombination,

and repair nucleases 6,7. A previous study showed that the C-terminal domain of PabNucS showed structural similarities

with RecB 8. However, the current results emphasized the similarities between EndoMS and type II restriction enzymes.

The type II restriction enzyme SgrAI was superposed with the C-terminal domain of EndoMS with an RMSD of 3.4 Å

for 91 residues; this was the most similar structure observed among known structures. Additionally, the restriction

enzymes FokI, AspBHI, Cfr10I, Bse634I, R.BspD6I, and BsoBI were detected. From this analysis, all of the detected

structures in complex with DNA were those of restriction enzymes (Figure S3). Therefore, these structures were

considered in modeling the EndoMS-dsDNA complex structure, as explained below.

The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu/) and ProOpDB

(http://operons.ibt.unam.mx/OperonPredictor/) were referenced in order to investigate genomic structures proximal to

the EndoMS gene 9,10. In ProOpDB, operons of the endoMS gene were searched, and operons consisting of the gene and

radA-related genes were conserved only in the archaeal Thermococcus genus. The genome structures of the predicted

operons were retrieved from the UCSC Archaeal Genome Browser for T. kodakaraensis KOD1, T. gammatolerans, T.

oonurience, T. sp. strain 4557, T. sibiricus, and T. barophilus MP (Figure S2) 11-15. STRING (http://string-db.org/) and

IntAct (http://www.ebi.ac.uk/intact/) databases were referenced for the experimentally detected protein-protein

interactions and for prediction of the functional relationships with EndoMS, respectively 16,17.

Knowledge-based modeling of the TkoEndoMS-dsDNA complex. According to the results of a database survey, two

of the EndoMS C-terminal domains were assumed to tether dsDNA in manner similar to that of restriction enzymes.

BsoBI was employed as the reference structure for the modeling because its catalytic domain exhibited the highest

similarity to that of EndoMS in terms of motif conservation and sizes of insertions/deletions (Figure S3a). The

N-terminal domains were thought to take part in a dimer formation. The C-terminal domain of PabNucS (residues

Ser126 to Pro233, PDB: 2VLD) was superposed onto the corresponding domains of BsoBI (PDB: 1DC1) in order to

construct a C-terminal domain dimer on dsDNA (Figure 6). The dsDNA was truncated, and nucleotides were replaced

according to the cocrystallized structure (G-T-mismatch-DNA1 in Figure S4a). The N-terminal domains of PabNucS

(residues Lys3 to Glu120) were dimerized as in the crystal structure of PabNucS. These N- and C-terminal models were

used as search models in molecular replacement, as detailed below.

Crystal structure analyses. The crystal structures of TkoEndoMS were determined with X-ray crystallography. The

mutant TkoEndoMS gene harboring Asp165Ala (D165A) inactive mutation was cloned, expressed, and purified as

described previously 18.

DNA-bound TkoEndoMS crystals were obtained under initial conditions using 80 mM sodium cacodylate

buffer (pH 6.5) containing 0.16 M calcium acetate, 14.4% (w/v) PEG8000, and 20% (w/v) glycerol for a 0.5-mL

reservoir and a mixture of 2 µL of the reservoir solution and 2 µL of protein solution in a 50 mM Tris-HCl (pH 8.0)

buffer containing 0.1 mM EDTA, 0.5 mM DDT, 10% (w/v) glycerol, 0.6 M NaCl, 200 µM TkoEndoMS dimer, and 200

µM dsDNA (T-G-mismatch-DNA1 in Figure S4a) for a hanging drop.

Crystals grew at 18°C to approximate maximum dimensions of 0.3 × 0.3 × 0.01 mm3 within a few weeks.

X-ray diffraction data were collected from loop-mounted crystals under cryogenic conditions with a CCD detector

Quantum315 (ADSC) at BL38B1, SMART6500 (Bruker AXS) at BL44XU in SPring-8 (Hyogo, Japan), or Quantum

270 (ADSC) at BL17A in Photon Factory (Tsukuba, Japan). The crystals were soaked for 10–30 s in a crystal growth

buffer containing 15% (v/v) 2-methyl-2,4-pentanediol (MPD) for cryoprotection. The diffraction images were

processed using the MOSFLM program 19,20.

The crystal structure was solved with the molecular replacement method using the Phaser-MR application

of PHENIX or MOLREP of CCP4 suites 21,22. A solution of the EndoMS-dsDNA crystal structure was initially

attempted using the crystal structure of PabNucS (PDB: 2VLD) for search models. TkoEndoMS and PabNucS

demonstrated 69.7% identity in amino acid sequences. PabNucS as dimers, monomers, or separated N-terminal and

C-terminal domains was used in both all-atom and Cβ models for molecular replacements. However, no promising

solution was obtained.

Since the database survey clearly indicated the similarities between EndoMS and type II restriction

enzymes, knowledge-based models of C-terminal domain dimer with dsDNA and N-terminal domain dimer were

examined as search models. The domains in the models were rendered into Cβ models and were applied for Phaser-MR.

Under these conditions, a solution that was reasonable in terms of crystal packing, R-values, and electron density map

was obtained. The initial R/free R factors of the model were 0.377/0.366, while those after three cycles of rigid body,

coordinates, and simulated annealing refinements were 0.355/0.386 for reflections between 25.0 and 2.4 Å resolution.

In the map after refinement, the electron densities for most of the side chains were clearly observed as residual densities

(Figure S5a).

The model refinements were conducted by using COOT and the phenix.refine application of PHENIX 21,23.

Because EndoMS was in homodimer form with two-fold symmetry, dsDNA might bind to the protein in two different

(opposing) directions. Since the dual-conformation was actually implied in the electron density map as a residual

density, the dsDNA model was duplicated and those in opposite directions were built in as alternative coordinates of

equal occupancy (Figures S5c, d, e, f, and g). In the initial refinement process, high residual densities were observed

proximal to many of the bases, where only one of the alternative dsDNA models was considered (an example electron

density is shown in Figure S5f). A high electron density proximal to Glu179 residues was interpreted to be a

magnesium ion (Figure S5b), because the atoms positioned on this density showed interatomic distances 2.15 Å in

average (s.d. 0.19 Å) to the coordinating oxygen atoms (values are those in the final models). Blobs of electron

densities observed between proteins and DNA were modeled in MPD, a cryoprotectant used in diffraction experiments,

because corresponding densities were not observed when data were collected without the cryoprotectant but in the

presence of a higher (~35%) glycerol concentration (data not shown). Final R /freeR-factors of 0.190/0.244 were

obtained for this crystal structure (Table I).

Structural analyses of the TkoEndoMS-dsDNA complex were also attempted for DNAs with different

mismatched base pairs (G-G, T-T, A-C, or T-C) or background sequences (DNA1, DNA2, or DNA3; Figure S4a).

DNA1, DNA2, and DNA3 have no consensus base except for those in the mismatch pairs. Crystallization experiments

were carried out using the same conditions as described above, and crystals were obtained for mismatch base pairs of

G-T, G-G, or T-T regardless of the background sequence. On the other hand, no crystals grew for DNAs containing

A-C or T-C mismatches, as well as normal A-T base pair. Although screening for crystallization conditions was

repeated, no crystals were obtained for complexes containing these DNAs. The structures of T-T-mismatch-DNA1,

G-G-mismatch-DNA1, G-T-mismatch-DNA2, and G-T-mismatch-DNA3 complexes were solved via molecular

replacement using the structure of the G-T-mismatch-DNA1 complex as a search model and were refined with the same

procedures as described above.

The crystals of apo TkoEndoMS grew in a stock solution (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.5 mM

DDT, 10% [w/v] glycerol, 0.6 M NaCl, and 445 µM TkoEndoMS) stored at 4°C. X-ray diffraction data were collected

with a CCD detector Quantum 270 at BL17A in the Photon Factory under cryogenic conditions and processed as

described above. Analysis of reflection data with the Xriage tool suggested that the crystal was twinned with an

operator (-h, l, k). Data collection from other apo-form crystals was carried out, and the crystals were always twinned

with fractions of 0.05–0.08. The crystal structure was solved with the molecular replacement method by separately

using N- and C-terminal domains of DNA-bound TkoEndoMS as search models. Model refinement was conducted as

mentioned above by applying the twin operator.

The quality of the models was evaluated by using the PROCHECK program 24. The parameters of dsDNA

were evaluated by using W3DNA web-tool, and compared with that of a canonical B-form DNA (PDB: 1BNA) (Table

S2) 25,26. The crystallographic parameters, data collection and refinement statistics, and PDB codes are summarized in

Table I. The molecular graphics were prepared with CHIMERA 27.

Evaluating Binding affinities of TkoEndoMS for various mismatch containing DNA.

Electrophoresis mobility shift assays (EMSA) were examined to quantify the DNA binding affinity of TkoEndoMS as

described previously 18. Various concentrations of TkoEndoMS were incubated with 5 nM 5'-Cy5-labeled dsDNA (15

bp) which had no mismatch base pair or contained single base-pair mismatches (G-G, G-T, T-T, A-A, T-C, A-C, A-A,

and C-C) in a reaction solution (20 mM Tris-HCl, pH 8.0, 6 mM (NH4)2SO4, 2 mM MgCl2, 100 mM NaCl, 0.1 mg/ml

BSA, and 0.1 % Triton X-100) at 37°C for 5 min. Relatively low concentrations of protein (0.5, 1, 2.5, 5, and 10 nM as

a dimer) were examined for preferred mismatches (G-G, G-T, and T-T), and higher concentrations were tested for

non-preferred mismatches (A-A, T-C, A-C, A-A, and C-C) (Figure S4c). The protein-DNA complexes were

fractionated by 8% native PAGE in 0.5 × TBE buffer. Typhoon Trio+ image analyzer and Image Quant TL software

(GE healthcare) were used to quantify the fluorescent signal in each DNA band. The apparent Kd values were

calculated based on a plotting of the rate of DNA binding versus the EndoMS concentration through non-linear

regression from three independent experiments.

Knowledge-based modeling of the TkoEndoMS-TkoPCNA-dsDNA complex. The structure of the

TkoEndoMS-TkoPCNA-dsDNA complex was modeled by assembling the structures from PDB as has been previously

applied for the P. furiosus DNA polymerase B-PCNA-dsDNA complex 28. Interface information for EndoMS-dsDNA,

PCNA-dsDNA, and EndoMS-PCNA was retrieved from the crystal structures determined in this study, the E. coli DNA

polymerase β subunit-DNA complex (PDB: 3BEP), and the P. furiosus PCNA-RFCL PIP-box complex (PDB: 1ISQ),

respectively 29,30.

First, the crystal structure of the TkoEndoMS-dsDNA complex was assembled onto that of the

PCNA-dsDNA complex by superposing the dsDNAs by gradually shifting the nucleotide segments for superposition

(Figure 6). The dsDNA on PCNA was extended in advance by 5 base pairs to the PIP-binding side of the trimer to

increase merging for DNA superposition. The superposed structure, in which EndoMS was placed in contact with

PCNA, but not so close as to cause steric hindrance, was selected for the next step. Second, the PIP-box of RFCL was

assembled to the model by superposing the PCNA subunits from the PCNA-dsDNA and PIP-box-PCNA complexes.

Finally, the C-terminal residues, which were disordered in the crystal structures, were added to the model to complete

the EndoMS structure by connecting the PIP-box to the C-terminal of the TkoEndoMS crystal structure (Arg240).

Electron microscopy and single particle image analysis. The stock solutions of purified TkoEndoMS (5 µM, i.e., 2.5

µM dimer), TkoPCNA (7.5 µM, i.e., 2.5 µM trimer), and synthetic dsDNA (2.5 µM, T-G-mismatch-DNA1’ in Figure

S4a) were mixed in a buffer solution (50 mM Tris-HCl, 0.1 mM EDTA, 0.5 mM DDT, 10% glycerol, 0.6 M NaCl, pH

8.0) and incubated at room temperature for 10 min.

The sample solution was diluted 20-fold a buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0), and 3 µL was

applied to a copper grid supporting a continuous thin-carbon film. The sample was left for 1 min and then stained with

three drops of 2% (w/v) uranyl acetate. Images of molecules were recorded with a TemCam-F216 CMOS camera

(TVIPS) with a pixel size of 3.0 Å/pixel (a total of 38 images) using a JEM1010 EM (JEOL), operated at an

accelerating voltage of 100 kV. A minimum dose system (MDS) was employed to reduce the electron radiation damage

of the sample.

Image analyses were carried out using the EMAN2 program suite 31. A total of 5,130 particle images of the

assumed TkoEndoMS-TkoPCNA-DNA complex were manually selected with the e2boxer tool. No filter was applied to

the individual images prior to image analysis. Alignment, classification, and averaging of the particle images were

performed using the e2refine2d tool. The average number of particles per class average was 65 (Figure S6c). The model

of the TkoEndoMS-TkoPCNA-DNA complex was used to generate density map projections with e2pdb2em and

e2proc2d tools for comparisons.

Tabl

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Supplementary Figure S1. Related to Figure 2. Alignment of putative EndoMS orthologs

Sequence alignment of the selected EndoMS homologs. The sequences are indicated as gene ID, genus, and species,

followed by higher order taxonomy, where Arc and Bac represent kingdom Archaea and Eubacteria, respectively. Eur,

Cre, Tha, Act, and DeiThe indicate the phyla Euryarchaeota, Crenarchaeota, Thaumarchaeota, Actinobacteria, and

Deinococcus-Thermus, respectively (species names are truncated in the alignment). Invariant amino acids are shaded,

and the residues consistent with the signature motif of type II restriction enzymes are underlined in the alignment.

a

b

Supplementary Figure S2. Related to Figure 2. Molecular phylogeny and genome structure of putative EndoMS

orthologs

(a) Molecular phylogeny of the selected EndoMS homologs. The sequences are indicated as gene ID, genus, and species,

followed by higher order taxonomy, where Arc and Bac represent kingdom Archaea and Eubacteria, respectively. Eur,

Cre, Tha, Act, and DeiThe indicate the phyla Euryarchaeota, Crenarchaeota, Thaumarchaeota, Actinobacteria, and

Deinococcus-Thermus, respectively (species names are truncated in the alignment). Invariant amino acids are shaded,

and the residues consistent with the signature motif of type II restriction enzymes are underlined in the alignment. (b)

Probable EndoMS operons in Thermococcus genomes are shown for T. kodakaraensis KOD1, T. gammatolerans, T.

oonurience, T. sp. strain 4557, T. sibiricus, and T. barophilus MP. Genes are shown as arrows, and possible operons are

boxed. Name of proteins encoded by the genes are abbreviated as MTAP (5'-methylthioadenosine phosphorylase),

MTAD (S-adenosylhomocysteine deaminase), SK channel (calcium-gated potassium channel), GK (glycerate kinase),

and CDC6 (cell division control protein 6).

Supplementary Figure S3. Related to Figure 2. Structures of EndoMS and type II restriction enzymes

(a) Amino acid sequence alignment of TkoEndoMS and type II restriction enzymes with similar catalytic domain

structures, namely, the type II restriction endonuclease SgrAI (3n78_A), type IIF restriction endonuclease Bse634I

(3v21_D), and restriction enzyme BsoBI (1dc1_D). The secondary structures of each protein are indicated with yellow

and green boxes for β-sheets and α-helices, respectively. The residues consistent with the signature motif of the type II

restriction enzyme are indicated with asterisks. (b) The quaternary structures of type II restriction enzymes and

TkoEndoMS are shown on top. The structures are superposed at the catalytic domains. The corresponding C-terminal

catalytic domains (green and pink), N-terminal dimerizing domains (blue and tan), and dsDNA (red and orange) are

colored for each protein. The superposition between catalytic domains of EndoMS (blue) and restriction enzymes

(green, tan, and sky blue for 3n78, 3v21, and 1dc1, respectively) are shown at the bottom. Catalytic residues of

EndoMS are shown as ball-and-stick models.

Supplementary Figure S4. Related to Figure 3. Sequence and affinity of DNAs for EndoMS substrates

(a) Nucleotide sequences of the dsDNAs cocrystallized in X-ray analyses or complexed in EM analyses with

TkoEndoMS. The mismatch bases are indicated as bases not in-line with the rest of the sequence, and the cleavage

positions are indicated with vertical lines. A comparison among the background sequences of DNA1, DNA2, and

DNA3 is shown on the right. The strands were selected such that base identities were as high as possible. (b)

Representative gel image for evaluating binding affinities of TkoEndoMS for DNA containing G-G, G-T, or T-T

mismatch, which are preferably recognized by the enzyme. The mismatched base pair and the protein concentrations

were shown above, and apparent Kd values were indicated below the gel image. (c) Representative gel image for

evaluating binding affinities of TkoEndoMS for DNA containing A-G, T-C, A-C, A-A, or C-C mismatches, which were

not bound by the enzyme.

Supplementary Figure S5. Related to Figure 3. DNA bound-structures of EndoMS

(a) The electron density map of TkoEndoMS in complex with G-T-mismatch-DNA1 contoured at 1.2 s is shown on the

model. The atoms are colored as in Figure 1a. (b) The omit (Fo - Fc) maps of TkoEndoMS in complex with

G-T-mismatch-DNA1 contoured at 5.0 s are shown on the model. The nucleotides at 5′ and 3′ sides of cleavage bond

and Mg2+ ion were omitted for the map shown in green. The mismatched nucleotides (only one of them is in sight) were

omitted for the map shown in magenta. (c) 2Fo - Fc map contoured at 1.2 s around the binding site of flipped bases

(G8A and T8B in alternative dsDNA models). (d) The omit (Fo - Fc) map of the same region as panel c contoured at

3.7 s, where T8B was omitted. (e) The omit (Fo - Fc) map of the same region as panel c contoured at 3.7 s, where G8A

was omitted. (f) The map of residual electron density contoured at 3.5 s around T9A-A7A or G9B-C7B pair. The

carbon atoms in the bases in alternative dsDNA model A (T9A-A7A) and B (G9B-C7B, which were not modeled at this

point) are shown in gray and white, respectively. (g) Alternative binding of dsDNA on TkoEndoMS. The same dsDNA

models were bound in opposite directions to each other on TkoEndoMS homo-dimer, and colored in red (alternative

model A) and yellow (alternative model B). (h) Superposition of TkoEndoMS-dsDNA complex structures. The proteins

in T-G-mismatch-DNA1, T-T-mismatch-DNA1, G-G-mismatch-DNA1, T-G-mismatch-DNA2, and

T-G-mismatch-DNA3 complexes are shown in light blue, tan, light green, pink, and gray, respectively. (i) A model of

dsDNA in B-form (PDB: 1BNA, colored in blue and sky) was superposed onto the bound dsDNA

(T-G-mismatch-DNA1, red and yellow) by ignoring the flipped out mismatched bases. The base-pair and base-step

parameters were compared between the dsDNAs and summarized in Table S2, which showed the dsDNA bound to

TkoEndoMS is in B-from as an overall.

Figure S6. Related to Figure 4. Electron microscopic images and class averages of the TkoEndoMS-TkoPCNA-

dsDNA complex

(a) An electron micrograph of the negatively stained TkoEndoMS-TkoPCNA-dsDNA complex (scale bar represents

100 nm). A large aggregate and several isolated particles (enclose in boxes with white lines) are noticeable. (b) Close

up image of the boxed area (black line) in panel a. (c) Two-dimensional class averages of the selected particles. The

size of each individual image is 19.2 × 19.2 nm2. The class averages indicated with white dots were compared with the

model projections in Figure 4c.

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