The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies

Abstract

The wall-loosening actions of b-expansins are known primarily from studies of EXPB1 extracted from maize (Zea mays) pollen. EXPB1 selectively loosens cell walls (CWs) of grasses, but its specific binding target is unknown. We characterized EXPB1 binding to sequentially extracted maize CWs, finding that the protein primarily binds glucuronoarabinoxylan (GAX), the major matrix polysaccharide in grass CWs. This binding is strongly reduced by salts, indicating that it is predominantly electrostatic in nature. For direct molecular evidence of EXPB1 binding, we conducted solid-state nuclear magnetic resonance experiments using paramagnetic relaxation enhancement (PRE), which is sensitive to distances between unpaired electrons and nuclei. By mixing 13 C-enriched maize CWs with EXPB1 functionalized with a Mn 2+ tag, we measured Mn 2+ -induced PRE. Strong 1 H and 13 C PREs were observed for the carboxyls of GAX, followed by more moderate PREs for carboxyl groups in homogalacturonan and rhamnogalacturonan-I, indicating that EXPB1 preferentially binds GAX. In contrast, no PRE was observed for cellulose, indicating very weak interaction of EXPB1 with cellulose. Dynamics experiments show that EXPB1 changes GAX mobility in a complex manner: the rigid fraction of GAX became more rigid upon EXPB1 binding while the dynamic fraction became more mobile. Combining these data with previous results, we propose that EXPB1 loosens grass CWs by disrupting noncovalent junctions between highly substituted GAX and GAX of low substitution, which binds cellulose. This study provides molecular evidence of b-expansin's target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.

Gradients in Wall Mechanics and Polysaccharides along Growing Inflorescence Stems

“…Many of these sharp signals can be assigned to pectins, such as the 108-ppm Ara C1 peak, the Rha and GalA signals at 101, 79, and 69 ppm, the 53.5-ppm methyl ester signal, and the 21-ppm acetyl signal of rhamnogalacturonan-I (RG-I). Three carbonyl peaks are observed at 172, 174, and 176 ppm and can be assigned to GalA methyl ester in homogalacturonan (HG), GalA acetyl, and GalA carboxylate in RG-I and HG, respectively (Wang et al, 2016a). The cellulose intensities in these inflorescence cell walls are much lower than those of seedling cell walls, as seen for the resolved interior cellulose C4 peak at 89 ppm and the C6 peak at 65 ppm (Dick-Pérez et al, 2011).…”

Diffuse Growth of Plant Cell Walls

“…Nevertheless, a recent study characterized a xylan in primary walls of Arabidopsis (Mortimer et al, 2015) that might selectively bind to the hydrophilic faces of microfibrils. Because b-expansin was recently shown to target xylans in grass cell walls (Wang et al, 2016a), it seems plausible that a-and b-expansins may loosen the connections between different faces of cellulose microfibrils in the wall.…”

Directed in vitro evolution of bacterial expansin BsEXLX1 for higher cellulose binding and its consequences for plant cell wall‐loosening activities

“…In summary, we supported and extended relationship between cellulose affinity and protein activity for the bacterial expansin BsEXLX1, which depends upon proper partitioning of protein within the plant cell wall. Enhanced binding to noncellulosic polysaccharides inhibited BsEXLX1 activity, in contrast to plant β‐expansins which preferentially bind and solubilize arabinoxylans in the course of loosening maize cell walls . These findings further emphasize that while BsEXLX1 shares homology with plant expansins, it has distinctive effects on cell walls compared with plant α‐ and β‐expansins.…”