Structural investigations of the glucan water dikinase 1 mechanism and flexibility

Abstract

Glucan-water-dikinase 1 (GWD1) plays an essential role in regulating starch metabolism in plants via O-6 phosphorylation of amylopectin. Here, we used biochemical characterization, AlphaFold2 modeling, X-ray crystallography and Small-Angle X-ray Scattering (SAXS) experiments to study its structure and catalytic mechanism. The protein is organized into five domains with two carbohydrate-binding modules (CBMs) at its N-terminal end followed by a central domain, whose structure was solved by X-ray crystallography in open and closed conformations. Next comes the domain carrying the catalytic histidine and the ATP-binding domain. We studied the spatial arrangement of the full enzyme and of several truncated forms by SAXS-driven modeling and identified a pivoting movement of the Histidine domain consistent with the enzymes autophosphorylation and subsequent phosphate transfer to a glucan. Our data suggest important residues at the domain interfaces that might assist catalysis and we hypothesize that the second CBM helps maintaining the catalytic domain close to the glucan chain for productive phosphate transfer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/704335v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1b860e5org.highwire.dtl.DTLVardef@1e172dcorg.highwire.dtl.DTLVardef@3c03edorg.highwire.dtl.DTLVardef@25c0d4_HPS_FORMAT_FIGEXP M_FIG C_FIG