Journal: Nature Communications

Article Title: The SAM domain of mouse SAMHD1 is critical for its activation and regulation

doi: 10.1038/s41467-017-02783-8

Figure Lengend Snippet: Crystal structure of full-length mSAMHD1 iso1 in complex with GTP and dGTP (2-Allo). a Surface representation of mSAMHD1 tetramer in three orthogonal views. Each subunit is shown in a different color (purple, orange, red, and green) with SAM domains highlighted. b Surface representation of the 2-Allo structure showing the allosteric binding pocket at the interface of three subunits (purple, red, and green). GTP and dGTP are shown with red sticks and the SAM-to-HD inter- and intra-subunit interactions are highlighted with selected interface residues shown in sticks. c Transparent surface representation of the SAM domain (lime green) capping the allosteric site

Article Snippet: Analytical size-exclusion chromatography Purified samples of mSAMHD1 protein (2 mg/ml, 200 μl) mixed with dGTP or GTP (4 mM final concentration) were applied to a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated in SAMHD1 buffer.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The SAM domain of mouse SAMHD1 is critical for its activation and regulation

doi: 10.1038/s41467-017-02783-8

Figure Lengend Snippet: Crystal structures of mSAMHD1 activation intermediates. a Surface and ribbon representations of the mSAMHD1 structures at the allosteric site. Nucleotides are shown in red sticks and each subunit is colored uniquely. Left, No-Allo structure with no nucleotides bound. Middle, 1-Allo structure with dGTP bound to Allo-site 1. Right, 2-Allo structure with GTP and dGTP bound to Allo-site 1 and Allo-site 2, respectively. b , c Putty representation comparing the No-Allo and 1-Allo mSAMHD1 structures ( b ) and comparing the 1-Allo and 2-Allo structures ( c ) in two orthogonal views. The structures are aligned by superposition of dimer A. RMSD values are represented using the color spectrum and the thickness of the coil. d , e Surface representation of the No-Allo (left), 1-Allo (middle), and 2-Allo (right) structures showing conformational differences. Red triangles and arrows highlight the separation between a pair of SAM domains (green and purple). Cartoon schematics of the No-Allo (left) and 2-Allo structures (right) indicate the swinging (top row) and compacting (bottom row) motions of dimer A′ toward dimer A. Double-headed arrows highlight the distances between SAM domains as the tetramer becomes more compact

Article Snippet: Analytical size-exclusion chromatography Purified samples of mSAMHD1 protein (2 mg/ml, 200 μl) mixed with dGTP or GTP (4 mM final concentration) were applied to a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated in SAMHD1 buffer.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: The SAM domain of mouse SAMHD1 is critical for its activation and regulation

doi: 10.1038/s41467-017-02783-8

Figure Lengend Snippet: The importance of SAM-to-HD interactions for mSAMHD1 oligomerization and activities. a , d Left, transparent surface representation of SAM-to-HD interface with important residues shown in sticks. Right, model of SAM-to-HD interface after mSAMHD1 residues are mutated to the corresponding hSAMHD1 residues. The red cross in a indicates a steric clash. b , e Left, SEC analysis of mSAMHD1 ( b ) or hSAMHD1 ( e ) variants before and after incubation with dGTP-α-S. Right, dNTPase assay for wild type and mutant mSAMHD1 proteins ( b ) or hSAMHD1 ( e ). Products were quantified by the malachite green assay. Each experiment was performed in triplicate. Error bars, s.d. c , f HIV-1 restriction by full-length mSAMHD1 iso1 ( c ) or hSAMHD1 ( f ) (WT and mutants) stably expressed in U937 cells after PMA differentiation. Immunoblotting (uncropped images in Supplementary Fig. 7 ) confirmed HA-tagged mSAMHD1 or hSAMHD1 expression. GAPDH was used as a loading control. Single-cycle HIV-1 infection of vector control cells was set as 100%. Each experiment was performed in triplicate. Error bars, s.d. *** p ≤ 0.0001

Article Snippet: Analytical size-exclusion chromatography Purified samples of mSAMHD1 protein (2 mg/ml, 200 μl) mixed with dGTP or GTP (4 mM final concentration) were applied to a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated in SAMHD1 buffer.

Techniques: Size-exclusion Chromatography, Incubation, Mutagenesis, Malachite Green Assay, Stable Transfection, Expressing, Infection, Plasmid Preparation