Two high-resolution crystal structures of potato 1,3-β-glucanase reveal subdomain flexibility with implications for substrate binding

Abstract

Endo-1,3-β-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for the hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubsituted -1,3-linked glucosyl residues. The plant enzymes belong to the glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. For endo-1,3-β-glucanase from Solanum tuberosum (potato, cultivar Désirée), X-ray diffraction data were collected to 1.40 and 1.26 Å resolution for two crystals which, despite a similar packing framework, represent two separate crystal forms. In particular, they differ in the Matthews coefficient and are consequently referred to as higher density (HD, 1.40 Å) and lower density (LD, 1.26 Å) forms. The general fold of the protein resembles that of other known plant endo-1,3--glucanases and is defined by a (β/α)8 barrel with an additional subdomain built around the C-terminal half of the barrel. The structures reveal high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with other GH17 endo-1,3-beta-glucanase structures reveals differences in the arrangement of the secondary structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate binding patterns. The crystal structures reveal an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His6-tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighboring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction.