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Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction.

Arnold LH, Groom HC, Kunzelmann S, Schwefel D, Caswell SJ, Ordonez P, Mann MC, Rueschenbaum S, Goldstone DC, Pennell S, Howell SA, Stoye JP, Webb M, Taylor IA, Bishop KN - PLoS Pathog. (2015)

Bottom Line: Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently.Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity.Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

View Article: PubMed Central - PubMed

Affiliation: The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London, United Kingdom.

ABSTRACT
SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

No MeSH data available.


Related in: MedlinePlus

Different G based nucleotides can be accommodated in the SAMHD1 allosteric site.(A) The contents and conformation of the allosteric sites for structures of SAMHD1(115–583)-ddGTP (top), SAMHD1(115–583[R164A])-dGTP (middle) and SAMHD1(115–626)-GTP (bottom) are shown. Nucleotides are shown in stick representation, SAMHD1 residues making contacts with the nucleotides are labelled. (B) SEC-MALLS analysis of SAMHD1(115–626) incubated with ddGTP/dATP(blue), dGTP/dATP (green) or GTP/dATP (orange). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks.
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ppat.1005194.g004: Different G based nucleotides can be accommodated in the SAMHD1 allosteric site.(A) The contents and conformation of the allosteric sites for structures of SAMHD1(115–583)-ddGTP (top), SAMHD1(115–583[R164A])-dGTP (middle) and SAMHD1(115–626)-GTP (bottom) are shown. Nucleotides are shown in stick representation, SAMHD1 residues making contacts with the nucleotides are labelled. (B) SEC-MALLS analysis of SAMHD1(115–626) incubated with ddGTP/dATP(blue), dGTP/dATP (green) or GTP/dATP (orange). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks.

Mentions: A comparison of the nucleotide configuration at the AL1 site for ddGTP, dGTP and GTP bound structures is shown in Fig 4A. The electron density that nucleotides were built into is presented in S6 Fig. The G base in all three structures makes the same hydrogen bonds with D137, Q142 and R145 of one monomer along with a stacking interaction with R451' of the opposing monomer. The basic side chains of K116 and R451' also make equivalent electrostatic interactions with the triphosphate of the bound nucleotide illustrating that the binding mode for each of the guanine nucleotides is broadly conserved. Therefore, the capacity of each G-based nucleotide to support tetramerisation when combined with dATP was assessed using SEC-MALLS (Fig 4B). These data show that all G-based nucleotides facilitate tetramer formation but to differing degrees in a rank order of GTP > dGTP > ddGTP. This supports the notion that in a cellular context, where the concentration of GTP is greater than that of any dNTP counterpart, tetramer formation is not limited by the availability of guanine-based nucleotides to occupy AL1, but instead by the availability and capacity of dNTPs to productively support tetramer formation through occupancy of AL2.


Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction.

Arnold LH, Groom HC, Kunzelmann S, Schwefel D, Caswell SJ, Ordonez P, Mann MC, Rueschenbaum S, Goldstone DC, Pennell S, Howell SA, Stoye JP, Webb M, Taylor IA, Bishop KN - PLoS Pathog. (2015)

Different G based nucleotides can be accommodated in the SAMHD1 allosteric site.(A) The contents and conformation of the allosteric sites for structures of SAMHD1(115–583)-ddGTP (top), SAMHD1(115–583[R164A])-dGTP (middle) and SAMHD1(115–626)-GTP (bottom) are shown. Nucleotides are shown in stick representation, SAMHD1 residues making contacts with the nucleotides are labelled. (B) SEC-MALLS analysis of SAMHD1(115–626) incubated with ddGTP/dATP(blue), dGTP/dATP (green) or GTP/dATP (orange). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592219&req=5

ppat.1005194.g004: Different G based nucleotides can be accommodated in the SAMHD1 allosteric site.(A) The contents and conformation of the allosteric sites for structures of SAMHD1(115–583)-ddGTP (top), SAMHD1(115–583[R164A])-dGTP (middle) and SAMHD1(115–626)-GTP (bottom) are shown. Nucleotides are shown in stick representation, SAMHD1 residues making contacts with the nucleotides are labelled. (B) SEC-MALLS analysis of SAMHD1(115–626) incubated with ddGTP/dATP(blue), dGTP/dATP (green) or GTP/dATP (orange). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks.
Mentions: A comparison of the nucleotide configuration at the AL1 site for ddGTP, dGTP and GTP bound structures is shown in Fig 4A. The electron density that nucleotides were built into is presented in S6 Fig. The G base in all three structures makes the same hydrogen bonds with D137, Q142 and R145 of one monomer along with a stacking interaction with R451' of the opposing monomer. The basic side chains of K116 and R451' also make equivalent electrostatic interactions with the triphosphate of the bound nucleotide illustrating that the binding mode for each of the guanine nucleotides is broadly conserved. Therefore, the capacity of each G-based nucleotide to support tetramerisation when combined with dATP was assessed using SEC-MALLS (Fig 4B). These data show that all G-based nucleotides facilitate tetramer formation but to differing degrees in a rank order of GTP > dGTP > ddGTP. This supports the notion that in a cellular context, where the concentration of GTP is greater than that of any dNTP counterpart, tetramer formation is not limited by the availability of guanine-based nucleotides to occupy AL1, but instead by the availability and capacity of dNTPs to productively support tetramer formation through occupancy of AL2.

Bottom Line: Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently.Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity.Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

View Article: PubMed Central - PubMed

Affiliation: The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London, United Kingdom.

ABSTRACT
SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

No MeSH data available.


Related in: MedlinePlus