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 8 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 3 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.