Fig. 6. CBM21 domain of PTG binds to the GYS1–GYG1 complex via the associated glucose chain.

a, Structural alignment of the AlphaFold predicted structure of the PTG(CBM21) domain against the starch-binding domain (SBD) from R. oryzae glucoamylase bound to maltotetraose and maltotriose at site I and site II, respectively. Panels show how site I and site II align with the putative GYS-binding motif and putative glycogen-binding motif. Both motifs are coloured green. Y203 and W246 labels are highlighted red. b, PTG(CBM21) was incubated with GYS1–GYG1^FL, GYS1–GYG1^pY195F or GYS1–GYG1^ΔCD. The ability of PTG to bind GYS1–GYG1 complexes was assessed by affinity pull-down, followed by SDS–PAGE (n = 4 technical repeats). c, PTG(CBM21) was incubated with GYG1 or GYG1^p.Y195F catalytic domain constructs, passed onto affinity resin and analysed by SDS–PAGE (n = 4 technical repeats). The GYG1 catalytic domain exists as a mixture of glucosylated states and runs at a higher apparent molecular weight in SDS–PAGE than GYG1^p.Y195F, which is nonglucosylated. d, Thermal shift analysis of PTG(CBM21) in the presence of various sugars and ligands (n = 4 technical repeats). P values between the apo and plus sugar samples were determined by two-tailed unpaired t-test. e, Thermal shift analysis of PTG(CBM21) wild type (labelled WT), PTG(CBM21)^p.Y203R variant (labelled Y203R) and PTG(CBM21)^p.W246R variant (labelled W246R) in the presence of increasing concentrations of maltoheptaose. Median melting temperatures (T_m) and standard deviations are shown (n = 3 technical repeats).

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