OVERCOMING OLIGOMERIC HETEROGENEITY TO DETERMINE THE FIRST HIGH-RESOLUTION STRUCTURE OF A GH173 GLYCOSIDE HYDROLASE

Glycoside hydrolases (GHs) are essential for the depolymerization of complex carbohydrates, enabling the breakdown of nature’s primary structural and energy-storage polysaccharides. Among them, GH173 remains an enigmatic family, with its molecular properties and biological function largely unexplored due to the absence of structural data. Here, we investigated the GH173 member BXY_21920 using biophysical and structural approaches. Size-exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering analyses revealed that BXY_21920 exists in multiple oligomeric states in solution, ranging from monomers to decamers, which interconvert in a redox-modulated dynamic equilibrium. The structural heterogeneity of the protein, which persisted even after size-exclusion chromatography, remained a major bottleneck for crystallization trials, frequently resulting in either failed attempts or poorly ordered crystals with low diffraction quality. To address this limitation, we performed limited proteolysis to identify structurally stable proteoforms suitable for crystallization. Optimization of the digestion conditions led to the growth of well-ordered crystals, allowing us to solve the monomeric structure of BXY_21920 at 2.1 Å resolution. The resulting structure lacked two loops adjacent to the active site, which were removed during proteolysis. To recover a more complete structural model, we rationally shifted the oligomeric equilibrium toward the monomer by adding TCEP just before crystallization. This strategy produced high-quality crystals and a 1.3 Å resolution structure that included the previously unresolved loops. Both structural models revealed a calcium-binding site and a conserved structural fold comprising a canonical catalytic (α/β)₈-barrel domain and a C-terminal β-sandwich domain. Notably, in the 1.3 Å structure, Cys119 was observed in three distinct rotamer conformations, each displaying extended electron density consistent with the formation of an intermolecular disulfide bond between Cys1 and Cys119 of neighboring protomers. Crystallographic symmetry analysis supported a trimeric arrangement, providing mechanistic insights into the protein’s oligomerization behavior. By overcoming the structural heterogeneity of BXY_21920, we determined the first high-resolution structure of a GH173 member. This study highlights the inherent complexity of structurally characterizing proteins that exist in heterogeneous and dynamic oligomeric states. Obtaining high-quality crystals in such cases requires careful optimization of experimental conditions.

This work was supported by FAPESP (2022/09386-8).