Expansin-like A binds to pectin via electrostatic forces and remodels the plant cell wall

Expansins are pivotal cell wall loosening proteins that facilitate turgor-driven extension of plant cell walls. Expansin-like A (EXLA) proteins represent a subfamily, but their interaction with polysaccharides remains poorly understood during primary cell wall growth, hindered by challenges in achieving active heterologous expression for in vitro analysis. Using an insect secretion-based expression system, we successfully expressed and purified EXLA proteins. Screening eight different polysaccharides showed that EXLAs exhibit a preference for binding to negatively charged polygalacturonic acid (PGA) and rhamnogalacturonan I (RG-I), pivotal components of pectin in the primary cell wall matrix. The crystal structure of EXLA1 was resolved at 2.5 Å resolution, revealing three crucial positively charged surfaces for pectin electrostatic interaction, and mutating these basic amino acids to alanine significantly reduced the binding ability. Moreover, recombinant EXLA1 promoted the extension of heat-inactivated cucumber hypocotyl walls under acidic conditions, indicating its intrinsic wall-loosening activity in vitro. EXLA1 overexpression resulted in a remodeled cell wall structure, suggesting EXLAs affect cell wall growth. These findings unveil EXLAs function during cell wall development by binding pectin through electrostatic interactions.