The target of β-expansin EXPB1 in maize cell walls from binding and solid-state NMR studies

Tuo Wang, Yuning Chen, Akira Tabuchi, Daniel J. Cosgrove, Mei Hong

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The wall-loosening actions of β-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 13C-enriched maize CWs with EXPB1 functionalized with a Mn2+ tag, we measured Mn2+-induced PRE. Strong 1H and 13C 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 β-expansin’s target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.

Original languageEnglish (US)
Pages (from-to)2107-2119
Number of pages13
JournalPlant physiology
Volume172
Issue number4
DOIs
StatePublished - Dec 2016

Fingerprint

expansins
Cell Wall
Zea mays
cell walls
corn
Poaceae
grasses
Cellulose
cellulose
Pollen
glucuronoarabinoxylan
nuclear magnetic resonance spectroscopy
binding proteins
Static Electricity
polysaccharides
Polysaccharides
electrons
pollen
Carrier Proteins
Magnetic Resonance Spectroscopy

All Science Journal Classification (ASJC) codes

  • Physiology
  • Genetics
  • Plant Science

Cite this

Wang, Tuo ; Chen, Yuning ; Tabuchi, Akira ; Cosgrove, Daniel J. ; Hong, Mei. / The target of β-expansin EXPB1 in maize cell walls from binding and solid-state NMR studies. In: Plant physiology. 2016 ; Vol. 172, No. 4. pp. 2107-2119.
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The target of β-expansin EXPB1 in maize cell walls from binding and solid-state NMR studies. / Wang, Tuo; Chen, Yuning; Tabuchi, Akira; Cosgrove, Daniel J.; Hong, Mei.

In: Plant physiology, Vol. 172, No. 4, 12.2016, p. 2107-2119.

Research output: Contribution to journalArticle

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AB - The wall-loosening actions of β-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 13C-enriched maize CWs with EXPB1 functionalized with a Mn2+ tag, we measured Mn2+-induced PRE. Strong 1H and 13C 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 β-expansin’s target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.

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