Structural analysis of β-L-arabinobiose-binding protein in the metabolic pathway of hydroxyproline-rich glycoproteins in Bifidobacterium longum

Masayuki Miyake, Tohru Terada (Corresponding Author), Michiko Shimokawa, Naohisa Sugimoto, Takatoshi Arakawa, Kentaro Shimizu, Kiyohiko Igarashi, Kiyotaka Fujita, Shinya Fushinobu (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

Abstract

Bifidobacterium longum is a symbiotic human gut bacterium that has a degradation system for β-arabinooligosaccharides, which are present in the hydroxyproline-rich glycoproteins of edible plants. Whereas microbial degradation systems for α-linked arabinofuranosyl carbohydrates have been extensively studied, little is understood about the degradation systems targeting β-linked arabinofuranosyl carbohydrates. We functionally and structurally analyzed a substrate-binding protein (SBP) of a putative ABC transporter (BLLJ_0208) in the β-arabinooligosaccharide degradation system. Thermal shift assays and isothermal titration calorimetry revealed that the SBP specifically bound Araf-β1,2-Araf (β-Ara2) with a Kd of 0.150 μm, but did not bind L-arabinose or methyl-β-Ara2. Therefore, the SBP was termed β-arabinobiose-binding protein (BABP). Crystal structures of BABP complexed with β-Ara2 were determined at resolutions of up to 1.78 Å. The findings showed that β-Ara2 was bound to BABP within a short tunnel between two lobes as an α-anomeric form at its reducing end. BABP forms extensive interactions with β-Ara2, and its binding mode was unique among SBPs. A molecular dynamics simulation revealed that the closed conformation of substrate-bound BABP is stable, whereas substrate-free form can adopt a fully open and two distinct semi-open states. The importer system specific for β-Ara2 may contribute to microbial survival in biological niches with limited amounts of digestible carbohydrates. Database: Atomic coordinates and structure factors (codes 6LCE and 6LCF) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Original languageEnglish
Pages (from-to)5114-5129
JournalFEBS Journal
Volume287
Issue number23
DOIs
Publication statusPublished - Dec 2020
MoE publication typeA1 Journal article-refereed

Funding

This study was supported by JSPS-KAKENHI (19H00929, 15H02443, 26660083, and 24380053 to S. F.) and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japanese Ministry of Education, Culture, Sports, and Technology (MEXT) and Japan Agency for Medical Research and Development (AMED) under Grant Number JP19am0101107. This study was also partially supported by JSPS-KAKENHI and a Grant-in-Aid for Innovative Areas from MEXT (19H03013 and 18H05494 to K.I.). K.I. thanks Business Finland (BF, Previously the Finnish Funding Agency for Innovation (TEKES)) for support from the Finland Distinguished Professor (FiDiPro) Programme, Advanced approaches for enzymatic biomass utilization and modification (BioAD).

Keywords

  • ABC transporter
  • human gut bacterium
  • isothermal titration calorimetry
  • molecular dynamics simulation
  • substrate-binding protein

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