High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP

Klemm, Bradley P.; Sikkema, Andrew P.; Hsu, Allen; Horng, James C.; Hall, T.M.T.; Borgnia, Mario J.; Schaaper, Roel M.

doi:10.1016/j.jbc.2022.102073

Cited by 4 publications

(10 citation statements)

References 67 publications

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“…In view of the proposed catalytic mechanism for Dgt ( SI Appendix , Fig. S8 ), which is proposed to be conserved with SAMHD1 ( 19 , 20 ), this alternate substrate conformation by itself is expected to be sufficient to inhibit the Dgt reaction by preventing the nucleophilic attack by the hydrolytic metal-bound water/hydroxide. However, additionally, R3′ forms an extended cation–π stacking interaction with the guanine ring and residues F391 A and H126 A ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The blue shape represents the active-site HD motif, and the purple and green circles are the proposed catalytic metal ions ( SI Appendix , Fig. S5 ) ( 20 ). Product release and any possible conformational change steps are excluded for simplicity.…”

Section: Discussionmentioning

confidence: 99%

“…All data were processed in RELION ( 39 ) using normal procedures ( SI Appendix , Table S2 and Fig. S10 ) using a workflow similar to what we described previously for the Leeuwenhoekiella blandensis dGTPase ( 20 ). Briefly, movie frames were aligned using MotionCor2 ( 40 ).…”

Section: Methodsmentioning

confidence: 99%

“…We recently adapted our previously published enzyme-coupled assay ( 51 ) to a 96-well plate format, as described for the L . blandensis dGTPase ( 20 ). Briefly, all reactions in this study were performed in a reaction buffer containing 100 mM Tris pH 8, 5 mM MgCl 2 , 125 µM MnCl 2 , and 5 mM inorganic phosphate pH 7.…”

Section: Methodsmentioning

confidence: 99%

“…All reactions were run in half-area, flat-bottom, ultraviolet-transparent Corning microplates (Sigma, CLS3679) with the change in absorbance at 297 nm monitored in real time in a SpectraMax M5 microplate reader (Molecular Devices). We used a previously determined experimental conversion factor to convert the change in absorbance to the product concentration in our plate assays ( 20 ). This conversion factor is the slope of the linear relationship of Δ A 297 endpoints from a dilution series of guanine reacted with XOD (1.5 × 10 −3 Δ A 297 /µM for the 40-µL reaction volume used in this study).…”

Section: Methodsmentioning

confidence: 99%

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Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase

Klemm

Singh

Smith

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2′-deoxyguanosine-5′-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host “arms race.” We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt–Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt–Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase

Klemm

Singh

Smith

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1

Orris,

Sung,

Bhat

et al. 2023

Nucleic Acids Research

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The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

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Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies

Orris

Sung

Bhat

et al. 2023

Preprint

View full text Add to dashboard Cite

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ~20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

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High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP

Cited by 4 publications

References 67 publications

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase

Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1

Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies

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