Figure 3. CHAD domains are specific polyP-binding modules.

(A) Quantitative GCI polyP-binding assay. Biotinylated polyP (average chain length is ∼100 Pi units) was immobilized on a streptavidin GCI chip, and the different CHAD domains were used as analytes (Fig S6). The yeast polyP polymerase Vtc4 and BSA were used as positive and negative controls, respectively. Shown are recorded sensograms (in red) with the respective fits (in black) and including table summaries of the derived association rate constant (k_a), dissociation rate constant (k_d), and dissociation constant (K_D). (B) Short-chain polyPs (average chain length ∼7 P_i units) can compete with CHAD domains for binding to the polyP-coated GCI chip. Shown are dose–response curves with derived IC₅₀ estimates. (C) Diadenosine pentaphosphate (AP5A) cannot compete with RcCHAD for binding to the polyP-coated GCI chip (right panel) as efficiently as polyP (left panel, average chain length ∼7 P_i units). Shown are sensograms of the association phase at indicated inhibitor concentration. (D) Sensograms for SsCHAD and RcCHAD reveal no significant interaction with biotinylated single-stranded DNA (54 nt, in orange) or single-stranded RNA (10 nt, in green). Biotinylated polyP (average chain length ∼100 P_i units, in red) is shown as a positive control. CtCHAD, however, binds biotinylated heparin. Shown are the recorded sensograms (in red) with the respective fits (in black) and including a table summary of the derived association rate constant (k_a), dissociation rate constant (k_d), and dissociation constant (K_D). (E) Phosphohydrolase activities of ygiF full-length 1–433 (FL) and ygif-TTM^1–200 (TTM) versus different phosphorylated substrates. Symbols represent raw data, lines indicate mean values, and error bars denote SD of three independent replicates. An SDS–PAGE analysis of the purified proteins is shown alongside. The theoretical molecular weight is ∼22.3 kD for TTM and ∼48.4 kD for FL.