Fig. 4: Binding stoichiometry in SAMHD1:oligonucleotide complexes.

a Size-exclusion chromatography (SEC) analysis of oligonucleotide:D311A SAMHD1 complexes formed with no GTP or dATP present. Absorbance measurements at two wavelengths, 280 and 495 nm, allow independent quantification of SAMHD1 and the oligonucleotide. The amount of oligonucleotide associated with the SAMHD1 dimer was determined by integrating the shaded area of the 495-nm chromatogram. The three dashed lines indicate the retention volumes of SAMHD1 monomer (M), dimer (D), and tetramer (T). b The same experiment as in (a) but performed in the presence of 50 μM GTP and 50 μM dATP in the running buffer and during incubation with the oligonucleotide. c Amounts of D311A SAMHD1-bound oligonucleotide, as determined by integrating the shaded areas of the 495-nm chromatograms in (a, b), plotted as a function of the total amount of the crude d(C*G*C*C*T) oligonucleotide added to SAMHD1. The left Y axis indicates absolute amounts and the right axis shows it as a fraction of the total amount of SAMHD1 (~5 μΜ) used in the titrations. d Analytical ultracentrifugation analysis of the d(C*G*C*C*T):D311A SAMHD1 complexes formed in the presence of GTP and dATP using 280- and 495-nm multiwavelength detection. Quantification of SAMHD1 and the oligonucleotide present in distinct complexes was performed by integrating the shaded areas of the plot. e Asymmetric oligonucleotide:SAMHD1 complexes formed as a result of the allosteric communication between distinct SAMHD1 monomers in the oligomer.