Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis

Morris, Elizabeth R.; Caswell, Sarah; Kunzelmann, Simone; Arnold, Laurence H.; Purkiss, A.; Kelly, Geoff; Taylor, Ian A.

doi:10.1038/s41467-020-16983-2

Cited by 28 publications

(61 citation statements)

References 86 publications

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“…The HD-domain superfamily fosters three different enzymatic classes: hydrolases, oxygenases, and lyases, with the hydrolases being the largest and best-characterized group. Hydrolases are further subdivided into (i) phosphatases, including dGTPases [38], RelA/SpoT [9,17], SAMHD1 [18,19], EF1143 [20,21], etc. and (ii) phosphodiesterases (PDEs), including exoribonucleases [39], PDEases [27][28][29][30][31][32][33][34], Cas proteins [23][24][25][26], and HD-GYPs [14,35,36,40] (Figure 1B).…”

Section: Characteristics and General Classification Of Hd-domain Proteinsmentioning

confidence: 99%

“…The main catalytic core is held to the protein polypeptide by four invariant residues, H167, H206, D207, and D311, which until recently was well accepted to bind a single metal ion [18,19]. A new structure, however, shows that the active site might be binuclear harboring a Fe-Mg center with the H233 coordinating the second metal ion (i.e., Mg) (Figure 3).…”

Section: Samhd1mentioning

confidence: 99%

“…A new structure, however, shows that the active site might be binuclear harboring a Fe-Mg center with the H233 coordinating the second metal ion (i.e., Mg) (Figure 3). The two metal ions are bridged by D207 and the substrate α-phosphate [19]. Two histidine residues vicinal to the active site, H210 and H215, form salt bridges with the phosphate oxygens [18], and together with R164 and H233, are important for catalytic activity [18].…”

Section: Samhd1mentioning

confidence: 99%

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The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries

et al. 2020

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The histidine–aspartate (HD)-domain protein superfamily contains metalloproteins that share common structural features but catalyze vastly different reactions ranging from oxygenation to hydrolysis. This chemical diversion is afforded by (i) their ability to coordinate most biologically relevant transition metals in mono-, di-, and trinuclear configurations, (ii) sequence insertions or the addition of supernumerary ligands to their active sites, (iii) auxiliary substrate specificity residues vicinal to the catalytic site, (iv) additional protein domains that allosterically regulate their activities or have catalytic and sensory roles, and (v) their ability to work with protein partners. More than 500 structures of HD-domain proteins are available to date that lay out unique structural features which may be indicative of function. In this respect, we describe the three known classes of HD-domain proteins (hydrolases, oxygenases, and lyases) and identify their apparent traits with the aim to portray differences in the molecular details responsible for their functional divergence and reconcile existing notions that will help assign functions to yet-to-be characterized proteins. The present review collects data that exemplify how nature tinkers with the HD-domain scaffold to afford different chemistries and provides insight into the factors that can selectively modulate catalysis.

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Section: Characteristics and General Classification Of Hd-domain Proteinsmentioning

confidence: 99%

Section: Samhd1mentioning

confidence: 99%

Section: Samhd1mentioning

confidence: 99%

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The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries

et al. 2020

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“…While we believe this unlikely to be the full explanation for the difference in glucocorticoid sensitivity based on other functional studies performed on these cells, future work will include generation of an untagged IK6 isoform to confirm these findings. While all IKZF1 engineered lesions result in relative resistance to most standard ALLdirected agents, we found a uniform increased sensitivity to the nucleoside analog cytarabine that appears to be a result of the reduced expression of SAMHD1, a dNTP/cytarabine-TP triphosphohydrolase, 48 which could be fully rescued with restoration of WT SAMHD1 protein levels. Cell line and primary patient data further corroborates that IKAROS regulates SAMHD1 expression.…”

Section: Discussionmentioning

confidence: 74%

Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities

Rogers

Gupta

Reyes³

et al. 2021

Blood Advances

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IKAROS family zinc finger 1 (IKZF1) alterations represent a diverse group of genetic lesions that are associated with an increased risk of relapse in B-lymphoblastic leukemia (B-ALL). Due to the heterogeneity of concomitant lesions, it remains unclear how IKZF1 abnormalities directly affect cell function and therapy resistance and whether their consideration as a prognostic indicator is valuable in improving outcome. We used CRISPR/Cas9 to engineer multiple panels of isogeneic lymphoid leukemia cell lines with a spectrum of IKZF1 lesions in order to measure changes in chemosensitivity, gene expression, cell cycle, and in vivo engraftment that can be linked to loss of IKAROS protein. IKZF1 knockout and heterozygous null cells displayed relative resistance to a number of common therapies for B-ALL including dexamethasone, asparaginase, and daunorubicin. Transcription profiling revealed a stem/myeloid cell-like phenotype and JAK/STAT upregulation after IKAROS loss. We also used a CRISPR homology-directed repair (HDR) strategy to knock-in the dominant-negative IK6 isoform into the endogenous locus and observed a similar drug resistance profile with the exception of retained dexamethasone sensitivity. Interestingly, IKZF1 knockout and IK6 knock-in cells both have significantly increased sensitivity to cytarabine, likely owing to marked downregulation of SAMHD1 after IKZF1 knockout. Both types of IKZF1 lesions decreased survival time of xenograft mice, with higher numbers of circulating blasts and increased organ infiltration. Given these findings, exact specification of IKZF1 status in patients may be a beneficial addition to risk stratification and could inform therapy.

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“…A structural model for the SAMHD1 monomer was extracted from the tetramer structure (PDB: 6TXC) (30). Normal mode analysis was carried out on the monomer chain in Rstudio using the package Bio3D (31).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation, and ssDNA binding kinetics

Orris

Huynh

Ammirati

et al. 2022

Preprint

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SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), enabling the enzyme to localize to stalled replication forks (RFs) and promote their restart. Since localization of a potent dNTPase at stalled RFs is not harmonious with DNA replication, we used a series of kinetic and thermodynamic measurements to explore a hypothesis where the combined effects of T592 phosphorylation and ssDNA binding serves as a dual switch to turn-off SAMHD1 dNTPase activity. We report that phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1. However, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (~15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial baseline dynamics in the C-terminal domain (which contains T592) which may be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.

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Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis

Cited by 28 publications

References 86 publications

The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries

The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries

Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities

Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation, and ssDNA binding kinetics

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