Structural Basis for Methyl Transfer by a Radical SAM Enzyme

Transcription

1 Supporting Online Material for Structural Basis for Methyl Transfer by a Radical SAM Enzyme Amie K. Boal, Tyler L. Grove, Monica I. McLaughlin, Neela H. Yennawar, Squire J. Booker,* Amy C. Rosenzweig* *To whom correspondence should be addressed. squire@psu.edu (S.J.B.); amyr@northwestern.edu (A.C.R.) This PDF file includes: Published 28 April 2011 on Science Express DOI: /science Materials and Methods Figs. S1 to S18 Table S1 References

2 Materials and Methods General crystallographic methods. All datasets were processed using the HKL2000 package (51) and solved by multiple anomalous dispersion (MAD) phasing using SHARP/autoSHARP (52, 53) or by molecular replacement using the program PHASER (54). Model building and refinement were performed with Coot and Refmac5, respectively (55, 56). Data collection and refinement statistics are shown in Table S1. Ramachandran plots were calculated with PROCHECK (57) and MolProbity (58). Diffraction-component precision index (DPI) errors were calculated with SFCHECK (59). Figures were prepared using PyMOL (60) and electrostatic surface potential calculations were performed with the PyMOL APBS plugin (49) at 150 mm monovalent ion concentration. All data were collected at the Life Sciences Collaborative Access Team (LS-CAT) and General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) beamlines at the Advanced Photon Source. RlmN structure. RlmN from Escherichia coli was prepared as described previously (7) from a construct encoding a C-terminal hexahistidine affinity tag that remains uncleaved after purification. All manipulations were carried out in a Coy anaerobic chamber. Protein solutions (100 mg/ml in 10 mm HEPES ph 7.5, 500 mm KCl, 10% glycerol, 5 mm DTT) were diluted 1:10 in 20 mm HEPES ph 7.6, allowed to stand overnight, and centrifuged at 10,000 g prior to crystallization. Yellow-brown rectangular prism-shaped crystals were obtained using the hanging drop vapor diffusion method at 20 C with 10% (w/v) PEG 6000, 5% (v/v) 2-methyl-2,4-pentanediol (MPD), 0.1 M HEPES ph 7.5 as a precipitant equilibrated against a 0.25 M LiCl well solution. Crystals were soaked in cryoprotectant solution (30% (w/v) glycerol, 10% (w/v) PEG

3 6000, 5% (v/v) MPD, 0.1 M HEPES ph 7.5) for less than 5 min, mounted on rayon loops, and flash cooled in liquid nitrogen. Diffraction data for MAD phasing were collected at the Fe absorption peak and a remote wavelength (Table S1). A high-resolution native data set was also collected. The peak and remote data sets were subjected individually to an initial round of autosharp that yielded the location of the iron-sulfur cluster. High resolution phase information was obtained by placing individual iron atoms within the cluster density using the Fe anomalous Fourier map, a model cluster, and the locations of the three cysteinyl ligands (61). Phasing with SHARP (53) (peak 2 and remote 2 datasets) with the eight iron sites (four sites per cluster, one cluster in each of the two molecules in the ASU) determined as above yielded good quality electron density maps after solvent flattening with SOLOMON (62). An initial model was built automatically using Buccaneer (63). Further adjustment manually with Coot (55) was aided by use of additional data from a second crystal phased as described above, but with a higher resolution native dataset included (peak 1, remote 1, and native 1 datasets). This map showed improved side chain density and was used during manual model building. Non-crystallographic symmetry restraints were used during model building but removed in the later stages of refinement. The final model was refined against the native 1 dataset and consists of residues and for chain A, residues and for chain B, eight iron atoms, eight sulfur atoms, one MPD molecule, and 366 water molecules. Electron density was not observed for residues in chains A and B. Ramachandran plots indicate that 99.9% of the residues are in the allowed and additionally allowed regions. The DPI error is Å.

4 RlmN+SAM structure. RlmN crystals obtained as described above were soaked in mother liquor containing 1 mm S-adenosyl-L-methionine (SAM) for 45 min in a Coy anaerobic chamber. Crystals were briefly transferred to cryoprotectant solution (30% (v/v) PEG 400, 10% (w/v) PEG 6000, 5% (v/v) MPD, 0.1 M HEPES ph 7.5), then mounted on rayon loops and flash frozen in liquid nitrogen. The structure was solved by molecular replacement using the coordinates of RlmN as a starting model. The final model consists of residues for chain A, residues for chain B, eight iron atoms, eight sulfur atoms, two SAM molecules, and 181 water molecules. Electron density was not observed beyond residue 349 in chain A. Ramachandran plots indicate that 100% of the residues are in the allowed and additionally allowed regions. The DPI error is Å.

5 Figure S1. The methylation reactions catalyzed by E. coli RlmN and S. aureus Cfr (7). RlmN methylates the C2 position of A2503. Cfr catalyzes methylation of C2 and C8, but C8 is the primary target.

6 Figure S2. The proposed mechanism for C2 methylation of A2503 by RlmN (7).

7 Figure S3. Secondary structure matching (SSM) superposition (PDBeFold server (64)) of the E. coli RlmN (purple) α 6 /β 6 core with the PFL-AE (PDB accession code 3CB8) core structure (30). Two views are shown, (A) and (B). The [4Fe-4S] cluster is shown as a space filling model in orange (iron) and yellow (sulfur) and the SAM cosubstrate is shown in stick format (green) and colored by atom type. The rmsd is 2.6 Å for 220 Cα atoms. The most significant differences between the two structures are found in the indicated loops connecting core secondary structure elements. The equivalent to loop C, located between β6 and α6, is shorter in RlmN and lacks the helical region. The loop between α1 and β2 is longer in RlmN and is located on the same side of the barrel as a loop in PFL-AE (not shown) that undergoes dramatic conformational change upon binding of a substrate analog (30). The structural differences in these loops may reflect differences in substrate specificity for the two enzymes.

8 Figure S4. Structure-based sequence alignment of the RlmN N-terminal domain with a representative HhH 2 structure (RuvA, PDB accession code 2C7Y). The fold is defined by a set of conserved hydrophobic residues contributed by each helix (bottom, black bars) and a GXG sequence conserved in the two hairpin regions (bottom, orange bars) (36). The paired GXG sequences typically confer sequence independent minor-groove recognition of B-form DNA through interaction with the phosphate backbone on both strands (36). In the RlmN domain, lack of conservation of the hairpin sequence and deviation from a pseudosymmetric arrangement of HhH motifs may result in specificity for an RNA structure. Similar deviations are observed in a domain found in DNA polymerase β which interacts with only one DNA strand (65).

9 Figure S5. The α1/β2 loop adopts different conformations and engages in crystal contacts. (A) Residues shown in stick format (RlmN structure, chain B). The β7 extension is shown in blue, the α 6 /β 6 core in purple, the β 1-3 extension in pink, and the N-terminal domain in green. In this molecule, the loop packs against a portion of the second molecule in the ASU, placing it close to the β 1-3 extension. (B) and (C) show an overlay of the three conformations observed in the four molecules from the two structures.

10 Figure S6. The C-terminal helix in the RlmN+SAM structure. (A) 2F o -F c (1.3σ) map of residues in chain B. (B) and (C) show interactions with a symmetry related molecule. The C- terminal helix also interacts in an intramolecular fashion with a short loop composed of residues (D-F) show zoomed-in views of hydrophobic (D) and hydrogen bonding interactions (E) with the symmetry related molecule (D and E) and residues from the same molecule (F). The side chain density is more pronounced for the residues that engage in specific hydrophobic or hydrogen bonding interactions. The intramolecular interactions shown in view (F) are not observed in the absence of SAM because the helix is shifted away from the core. Notably, the residues that comprise this helix are absent in Cfr.

11 Figure S7. The iron-sulfur cluster in RlmN and RlmN+SAM. 2F o -F c electron density map (1.5σ) for the side chains of residues 125, 129, and 132 within the CX 3 CX 2 C motif, the [4Fe-4S] cluster, and the SAM cofactor in the RlmN+SAM structure. The SAM methionine sulfur atom is located 3.7 Å from the two nearest sulfur atoms in the [4Fe-4S] cluster and 3.2 Å from the unique iron site. These distances are consistent with other structurally characterized radical SAM enzymes (39). In the inset, an Fe edge anomalous difference electron density map (9.0σ) for the RlmN structure shows full occupancy of all four iron atoms in the cluster.

12 Figure S8. A hydrogen bonding network between SAM (shown as sticks, green), the α 6 /β 6 core, residues (colored as in Fig. 1), and ordered solvent (red spheres). Additionally, Glu 105 forms hydrogen bonds to the backbone of the linker region. These interactions may trigger ordering of residues upon SAM binding. The use of water-mediated contacts may be an especially important feature of this network, allowing for controlled repositioning of the loop within the active site.

13 Figure S9. Overlay of the RlmN (light gray) and RlmN+SAM structures (colored as in Fig. 1). (A) A view of the entire structure. Areas that undergo large changes are indicated by black arrows. The C-terminal helix (blue) experiences a significant positional shift due to ordering of residues (B) A zoomed-in view of the SAM binding site. Conserved residue Asn 312 shifts 2.5 Å (Cα) to fold over the face of the SAM adenine moiety. (C) Loops near the active site shift position or become ordered (black arrows) upon SAM binding resulting in a less solvent exposed active site at the C-terminal end of the barrel. Similar ordering phenomena and conformational changes are observed upon substrate binding in other RS enzymes (30) and are proposed to prevent unwanted chemistry such as abortive cleavage of SAM.

14 Figure S10. 2F o -F c electron density map (1.0σ) for residues in the RlmN+SAM structure. Weak electron density is observed for two residues (Ala 353 and Gly 360), labeled with asterisks, suggesting conformational flexibility.

15 Figure S11. A stereoview of the active site of RlmN.

16 Figure S12. Active site overlay of RlmN+SAM with RlmN (white). Cys 118 changes rotamer upon SAM binding, inducing a flip in the Met 176-Gly 177 peptide bond (or vice versa). This may result in stabilization of the SAM-bound state and favorable positioning of the Met 176 backbone carbonyl and Cys 118 side chain for various steps in the reaction pathway. The location of the Met 176 side chain methyl group, 4 Å from the site of 5 -da formation, may be important in controlling access to the reactive site.

17 Figure S13. Methyl acquistion by Cys 355 may occur via SAM bound to the [4Fe-4S] cluster. (A) The mcys 355 sulfur atom is positioned favorably to be deprotonated by nearby strictly conserved residue Glu 105 (perhaps via a local water network) and to subsequently attack the carbon atom of the SAM methyl substituent. (B) Local hydrogen bonding interactions may be altered in the presence of unmethylated Cys 355 to further facilitate methyl transfer or downstream reaction steps.

18 Figure S14. Comparison of the RlmN+SAM structure (colored as in Fig. 1) and the PFL- AE+peptide substrate (yellow) structure (30) (PDB accession code 3CB8). (A) Side-by-side view showing that the general location of the peptide substrate in the PFL-AE complex structure is similar to that of residues in RlmN+SAM. This observation is notable since these enzymes both form a protein centered radical in these regions upon 5 -da generation in the active site. (B) Side-by-side view showing that the PFL peptide glycyl radical site (Gly 734) (right) is shifted toward the 5 carbon (*) of the SAM cofactor compared to the position of the methyl substituent on mcys 355 in RlmN+SAM (left). An important structural feature of the loop may be the exposure of backbone carbonyls (such as that of Gly 734, shown) to interact with the SAM cosubstrate. Similar interactions may guide positioning of mcys 355 in RlmN upon substrate binding. (C) A manual alignment of the backbone atoms in the PFL-AE substrate and RlmN residues shows a similar finger loop structure perhaps defined by a potential hydrogen bonding interaction indicated by the gray dashed line. The site of radical formation in RlmN (mcys 355) does not align here with the glycyl radical site in PFL (Gly 734). It aligns with an adjacent serine, suggesting the site of reactivity is not controlled by the sequence of the loop but instead by its precise location in the active site. In each system, distal structural elements in the enzyme/substrate complex may ultimately control the position of the loop.

19 Figure S15. An additional view in the comparison of the electrostatic surface potential (A) in RlmN to a sequence conservation map from pairwise alignment of E. coli RlmN and S. aureus Cfr (B) as shown in Figure 3.

20 Figure S16. Sequence conservation near the active site in a pairwise alignment of E. coli RlmN and S. aureus Cfr. (A) shows strictly conserved residues in orange, neutral substitutions in tan, and variable regions in white. The surface near mcys 355 and Cys 118, residues identified as mechanistically critical, is highly conserved in both enzymes. (B) is colored as in Fig. 1. Specific residues substituted in Cfr are shown in stick format and labeled in black. Several relatively conservative substitions (Thr, Val in RlmN to Ser in Cfr) may subtly alter hydrogen bonding patterns to promote substrate conformational changes. A salt bridge between conserved residue Arg 344 and Glu 278 (Ala in Cfr) could be an important factor in substrate orientation within the RlmN active site.

21 Figure S17. Surface map (ConSURF server) (66) of sequence conservation among the entire RlmN/Cfr family of enzymes (700 sequences) (8). Views in (A), (B), and (C) are as shown in Figs. 3A-D, and S15.

22 Figure S18. Overlay of a Cfr homology model (I-TASSER server) (67) (tan) onto the RlmN+SAM structure (colored as in Fig. 1). Brackets denote large structural insertions found in RlmN.

23 Table S1. Data collection and refinement statistics. Data collection Wavelength (Å) RlmN Native 1 1 RlmN Peak 1 1 RlmN Remote 1 1 RlmN Peak 2 2 RlmN RlmN+SAM 1 Remote Space group C222 1 P Cell dimensions a, b, c (Å) 72.15, 80.41, , 80.46, ( ) (0.412) 72.19, 80.42, ( ) (0.488) 72.14, 80.33, ( ) (0.645) 72.24, 80.43, ( ) (0.688) 55.18, 55.62, Resolution (Å) ( ) ( ) R sym or R merge (0.529) (0.507) <I /σi> 23.9 (2.1) 26.0 (5.1) 20.3 (2.4) 32.2 (7.1) 23.6 (4.3) 16.5 (2.0) Completeness 99.3 (92.3) (99.7) 98.1 (82.9) (95.1) (%) (99.8) (99.9) Redundancy 7.0 (4.4) 13.2 (7.8) 12.3 (4.1) 28.5 (24.5) 14.1 (11.9) 4.9 (3.6) Refinement Resolution (Å) No. reflections R work / R free 0.213/ /0.242 No. atoms Protein Ligand/ion Water B-factors (Å 2 ) Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles ( ) Data collected at LS-CAT beamlines 21-ID-D, 21-ID-G, 21-ID-F 2 Data collected at GM/CA-CAT beamline 23-ID-D

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