Supplementary Fig. S1. SAMHD1c has a more potent dntpase activity than. SAMHD1c. Purified recombinant SAMHD1c and SAMHD1c proteins (with
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1 Supplementary Fig. S1. SAMHD1c has a more potent dntpase activity than SAMHD1c. Purified recombinant SAMHD1c and SAMHD1c proteins (with concentration of 800nM) were incubated with 1mM dgtp for the indicated times at 25 C. The amount of dg generated at each indicated time point is shown, as detected by the absorbance at A260nm.
2 Supplementary Fig. S2. Electrostatic potential surface maps of SAMHD1. (a) Blue indicates positive charge, and red indicates negative charge. The allosteric sites (in three circles) have a high degree of positive charge. (b) and (c) are rotated around the vertical axis by 180 degrees relative to (a). Interestingly, the active site has both positive and negative charge.
3 Supplementary Fig. S3. Topology diagram of SAMHD.1 SAMHD1 contains two additional N-terminal β-sheets (green).
4 a Allosteric site: dgtp-1 b Allosteric site: dgtp-2 Supplementary Fig. S4. Schematic diagram of protein-ligand interactions in the allosteric site. Analyzed by Ligplot 48. Blue bonds represent dntp ligands, and yellow bonds represent the SAMHD1 protein. Hydrogen bonds and coordination bonds are indicated by green dashed lines. Hydrophobic contacts with dntp are shown as yellow semicircles with radiating spokes. N2 of dgtp2 has no interaction with any of the amino acids in the allosteric site and datp can also be tolerated.
5 c Supplementary Fig. S5. Electron density maps for dntps in the allosteric sites at 1.8 Å resolution. Grey: 2Fo Fc densities at +1.5σ level; Red and green: Fo Fc densities at -3σ level and +3σ level, respectively. Green sphere: Mg ion. For Site2, when it is assigned as dgtp(shown in a), red negative Fo-Fc densities are observed at N2 atom. When it is assigned as datp (shown in b), green positive Fo-Fc densities are observed near C2 atom. So Site2 of the allosteric site should be occupied by a mixture of dgtp and datp. (c) A stereo image of a portion of the electron density map for the allosteric site of SAMHD1 (2Fo Fc map contoured at +1.0σ level).
6 Supplementary Fig. S6. Schematic diagram of protein-ligand interactions in the active site. Analyzed by Ligplot. Blue bonds represent a dgtp substrate, and yellow bonds represent SamHD1. H bonds are shown as green dashed lines. Hydrophobic contacts are shown as yellow semicircles with radiating spokes. The H-bond marked by a red star does not exist in the datp substrate.
7 Supplementary Fig. S7. A detailed comparison of active sites from our structure and 3U1N. The regions connecting the active site and the allosteric site are shown in red (aa ) and purple (aa ). 3U1N is in beige.
8 Supplementary Fig. S8. Comparison with 3U1N. (a) The dimer comparison. The 3U1N dimer is shown in beige. The interactions are similar to each other, except for the N-terminal shown in the red circle, which binds to an allosteric site in our structure. The arrow indicates a movement of the C-terminal region. (b and c) Two views of the monomer comparison, rotated by 90. Cyan, blue, and green indicate the three domains of our structure. Beige indicates 3U1N. It shows that the relative position of the N-terminal (cyan and blue) and C-terminal regions is a major difference between the two structures.
9 Supplementary Fig. S9. Effects of SAMHD1 variants on SIVmac Vpx-induced degradation. (a) Immunoblot of SAMHD1 WT and variants in the presence and absence of Vpx. HA-tagged SAMHD1 constructs were co-expressed with Flag-tagged Vpx or empty vector in HEK293T cells. Cell extracts were harvested 48 h later and analyzed by SDS-PAGE, followed by immunoblotting to detect SAMHD1-HA, SAMHD1c -HA, SAMHD1c-HA, and Flag-Vpx. Tubulin was used as the loading control. Vpx-induced degradation of SAMHD1-HA and SAMHD1c -HA was detected (lanes 1-6). In contrast, Vpx-induced degradation of SAMHD1c-HA was not detected (lanes 7-9). (b) The bar graph shows the percentages of the relative band intensities for SAMHD1 in the presence of Vpx vs. in the absence of Vpx (set to 100%). The error bars indicate the S.D. of five independent experiments.
10 Supplementary Fig. S10. Modulation of human LINE-1 retrotransposition by SAMHD1 tetramer. (a) Schematic of 99 PUR RPS EGFP (LINE-1) and JM111 EGFP (JM111), which have been previously described. The 99 PUR RPS EGFP contains combined promoters from both CMV and the 5 -UTR of the LINE-1. An antisense cassette of EGFP containing an inserted intron sequence was cloned near the 3' end of the L1 3' UTR. JM111 contains a double mutation of R261A/R262A in ORF1, which has been determined to abolish the retrotransposition activity of LINE- 1. (b). Representative flow chart of LINE-1 retrotransposition assay. The negative control JM111, containing mutations in ORF1, gave few GFP-positive cells and was used for flow cytometry gating. VR1012 was the empty vector used for SAMHD1 expression. SAMHD1-expressing plasmids (SAMHD1c, SAMHD1c D137A, or SAMHD1c) were co-transfected with LINE-1 into HEK293T cells. GFP-positive cells were determined by flow cytometry 5 days after transfection. (c) Tetramer formation of SAMHD1 is required for LINE-1 inhibition. Plasmids expressing SAMHD1c, SAMHD1c D137A, or SAMHD1c were co-transfected with LINE-1 into HEK293T cells. VR1012 was used as negative control for SAMHD1 expression,
11 while JM111 as negative control for LINE-1. EGFP-positive cells were determined by flow cytometry 5 days after transfection. All the data in this figure are representative of at least three independent experiments. The error bars indicated the S.D. of three replicates within one experiment. Student s t test was performed with Microsoft Excel to calculate p-values.
12 Supplementary Fig. S11. Modulation of mouse LINE-1 retrotransposition by SAMHD1 variants. (a) Schematics of the synthetic mouse LINE-1 construct used in this study. The mouse LINE-1 construct is based on native murine LINE-1 sequence, with theorf1p and ORF2p coding region optimized for expression in human cells. Also, it lacks part of the 3'-UTR sequence, which is replaced with a SV40pA signal. (b) SAMHD1c' has a higher potency against murine LINE-1 than does SAMHD1c. The negative control JM111, containing mutations in ORF1, showed few GFP-positive cells and was used for flow cytometry gating. VR1012 was the empty vector used for SAMHD1 expression. SAMHD1-expressing plasmids (SAMHD1c or SAMHD1c) were co-transfected with LINE-1 into HEK293T cells. GFP-positive cells were determined by flow cytometry 5 days after transfection. The expression of HA-tagged SAMHD1-related proteins was examined by immunoblot analysis using an anti-ha antibody. Tubulin was used as the cellular protein loading control. All the data in this figure are representative of at least three independent experiments. The error bars indicated the S.D. of three replicates within one experiment. Student s t test was performed with Microsoft Excel to calculate p-
13 values. (c) Representative flow cytometry data.
14 Supplementary Fig. S12. A representation of the mutations found in AGS patients. Labeled residues in sticks and red color indicate the AGS-related mutations.
15 Supplementary Fig. S13: Full scan for western blotting result shown in Supplementary Figure S9a.
16 Supplementary Fig. S13 (cont.). Full scan for western blotting result shown in Supplementary Figure S10c.
17 Supplementary Fig. S13 (cont.). Full scan for western blotting result shown in Supplementary Figure S11b. The lane next to the marker is irrelevant to this study.
18 Supplementary Table S1. Potential impact of AGS-related mutations on SAMHD1 H123P R143H R145Q H167Y I201N/M254V G209S L369S Located in β2 and forms an H bond with D314. D314 also forms an H bond with H167, which is involved in Zn coordination. H123P may affect Zn binding in the active site and β2 structure in the allosteric site. R143H mutation loses interaction with H210 and affects amino acid interactions in the active site. Furthermore, R143 is next to Q142 and R145, which are important for dgtp binding. R143H may affect allosteric site function. R145 directly binds dgtp in the allosteric site. H167 coordinates Zn in the active site, and mutation may affect protein folding. I201 is close to H206 and D207, which are important for Zn coordination. I201 also faces M254, which is in a hydrophobic pocket. I201N and M254V may affect protein folding. Next to D207, which is involved in Zn binding. G209S would affect D207 and Zn binding and the active site structure because of its longer side chain. L369 is located between the active site and allosteric site. L369S will create an interaction with Y374 and affect active site function. M385V M385 is in a hydrophobic pocket created by I444, I448, L453, F454 and supports loop Loop interacts with allosteric site.
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